AU781500B2 - Method for enhancing cellulose and modifying lignin biosynthesis in plants - Google Patents

Description

METHOD FOR ENHANCING CELLULOSE AND MODIFYING LIGNIN BIOSYNTHESIS IN PLANTS FIELD OF THE INVENTION This invention relates to polynucleotide molecules encoding cellulose synthase, promoters of cellulose synthase and cellulose synthase polypeptides, methods for genetically altering cellulose lignin biosynthesis, and methods for improving strength properties of juvenile wood and fiber in trees. The invention further relates to methods for identifying regulatory elements in a cellulose synthase promoter and transcription factors that bind to such regulatory elements, and to methods for augmenting expression of polynucleotides operably linked to a cellulose synthase promoter.

SoBACKGROUND OF THE INVENTION o* In this specification where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of S 15 knowledge or any combination thereof was at the priority date publicly available, known to •the public, part of the common general knowledge or known to be relevant to an attempt to solve any problem with which this specification is concerned.

Lignin and cellulose are the two major building blocks of plan cell walls that provide mechanical strength and rigidity. In plants, and especially in trees, these two organic materials exist in a dynamic equilibrium conferring mechanical strength, water transporting ability and protection from biotic and abiotic environmental stresses.

Normally, oven-dry wood contains 30 to 50 cellulose, 20 to 30 lignin and 20 to 30 hemicellulose (Higuchi, 1997).

Proportions of lignin and cellulose are known to change with variation in the natural environment. For example, during the development of compression wood in conifers, the percentage of lignin increases from 30 to 40 and cellulose content proportionally decreases from 40 to 30 (Timmell, 1986). Conversely, in angiosperm tension wood the percentage of cellulose increases from 30 to 40 while lignin content decreases from 30 to 20 (Timmell, 1986).

It was recently discovered that the genetic down-regulation of a key tissue-specific enzyme from the lignin biosynthesis pathway, 4CL, results in reduction of lignin 7718456.01 WO 00/71670 PCT/US00/13637 -2content by up to 45% in transgenic aspen trees (Hu et al., 1999). This down-regulation is also associated with a 15% increase in the cellulose content. If the converse were true, i.e., that increasing cellulose content by genetic up-regulation of cellulose biosynthesis results in reduction of lignin content, then the pulp yield could be increased. This would allow tremendous savings in chemical and energy costs during pulping because, for example, lignin must be degraded and removed during the pulping process.

Cellulose is a linear glucan consisting of P-D-1,4-linked glucose residues.

It is formed by a cellulose synthase enzyme which catalyzes assembly of UDP-glucose units in plasma membrane complexes known as "particle rosettes" (Delmer and Amor, 1995). Cellulose synthase is thought to be anchored to the membrane by eight transmembrane binding domains to form the basis of the cellulose biosynthesis machinery in the plant cell wall (Pear et al., 1996).

In higher plants, the glucan chains in cellulose microfibrils of primary and secondary cell walls are different in their degree of polymerization (Brown et al.. 1996).

For example, secondary cell walls are known to contain cellulose having a high degree of polymerization, while in primary cell walls the degree of polymerization is lower. In another example, woody cell walls suffering from tension stress produce tension wood on the upper side of a bent angiosperm tree in response to the stress. In these cells, there are elevated quantities of cellulose which have very high crystallinity. The formation of highly crystalline cellulose is important to obtain a higher tensile strength of the wood fiber. Woody cell walls located at the under side of the same stem experience a compression stress, but do not produce highly crystalline cellulose. Such variation in the degree of polymerization in cell walls during development is believed to be due to different types of cellulose synthases for organizing glucose units into different paracrystalline arrays (Haigler and Blanton, 1996). Therefore, it would be advantageous to determine the molecular basis for the synthesis of highly crystalline cellulose so that higher yields of wood pulp having superior strength properties can be obtained from transgenic trees. Production of highly crystalline cellulose in transgenic trees would also markedly improve the mechanical strength properties of juvenile wood formed in normal trees. This would be a great benefit to the industry because juvenile wood is generally undesirable for solid wood applications because it has inferior mechanical properties.

Since the deposition of cellulose and lignin in trees is regulated in a compensatory fashion, genetic augmentation of cellulose biosynthesis might have a repressive effect on lignin deposition. Since the degree of polymerization and crystallinity may depend upon the type of cellulose synthase incorporated in the cellulose biosynthesis machinery, the expression of heterologous cellulose synthase or a UDP-glucose binding region thereof sweetgum protein expression in loblolly pine), could increase the quality of cellulose in transgenic plants. Over-expression of a heterologous cellulose WO 00/71670 PCTIUSOO/13637 -3synthase may also increase cellulose quantity in transgenic plants. Thus, genetic engineering of cellulose biosynthesis can provide a strategy to augment cellulose quality and quantity, while reducing lignin content in transgenic plants.

A better understanding of the biochemical processes that lead to wood formation would enable the pulp and paper industries to more effectively use genetic engineering as a tool to meet the increasing demands for wood from a decreasing production area. With this objective, many xylem-specific genes, including most lignin biosynthesis genes, have been isolated from developing xylem tissues of various plants including tree species (Ye and Varner, 1993; Fukuda, 1996; Whetten et al., 1998). Genes regulating cellulose biosynthesis in crop plants (Pear et al., 1996 and Arioli et al., 1998), versus in trees, have also been isolated. However, isolation of tree genes which are directly involved in cellulose biosynthesis has remained a great challenge.

For more than 30 years, no gene encoding higher plant cellulose synthase (CelA) was identified. Recently, Pear et al. (1996) isolated the first putative higher plant CelA cDNA, GhCelA (GenBank No. GHU58283), by searching for UDP-glucose binding sequences in a cDNA library prepared from cotton fibers having active secondary wall cellulose synthesis. GhCelA was considered to encode a cellulose synthase catalytic subunit because it is highly expressed in cotton fibers, actively synthesizes secondary wall cellulose, contains eight transmembrane domains, binds UDP-glucose, and contains two other domains unique to plants.

Recently, Arioli et al. (1998) cloned a CelA homolog, RSW1 (radial swelling) (GenBank No. AF027172), from Arabidopsis by chromosome walking to a defective locus of a temperature sensitive cellulose-deficient mutant. Complementation of the rswl mutant with a wild type full-length genomic RSW1 clone restored the normal phenotype. This complementation provided the first genetic proof that a plant CelA gene encodes a catalytic subunit of cellulose synthase and functions in the biosynthesis of cellulose microfibrils. The full-length Arabidopsis RSW1 represents the only known, currently available cellulose synthase cDNA available for further elucidating cellulose biosynthesis in transgenic systems (Wu et al., 1998).

The discovery of the RSW1 gene substantiated the belief that the assembly of a cellulose synthase into the plasma membrane is required for functional cellulose biosynthetic machinery and for manufacturing crystalline cellulose microfibrils in plant cell walls. Most significantly, a single CelA gene, e.g. RSW1, is sufficient for the biosynthesis of cellulose microfibrils in plants, e.g. Arabidopsis. Thus, RSW1 is a prime target for engineering augmented cellulose formation in transgenic plants.

Since many of society's fiber, chemical and energy demands are met through the industrial-scale production of cellulose from wood, genetic engineering of the cellulose biosynthesis machinery in trees could produce higher pulp yields. This would WO 00/71670 PCT/USOO/13637 -4allow greater returns on investment by pulp and paper industries. Therefore, it would be advantageous to isolate and characterize genes from trees that are involved in cellulose biosynthesis in order to improve the properties of wood.

SUMMARY OF THE INVENTION The present invention relates to polynucleotides comprising a nucleotide sequence that encodes a cellulose synthase, regulatory sequences, including a stressinducible promoter, of the cellulose synthase, a cellulose synthase protein or a functional domain thereof and methods for augmenting cellulose biosynthesis in plants.

Thus, in one aspect, the invention provides a polynucleotide comprising a sequence that encodes a cellulose synthase, or a polynucleotide fragment thereof, the fragment encoding a functional domain of cellulose synthase, such as a UDP-glucose binding domain. The invention also provides a cellulose synthase or a functional domain or fragment thereof, including a UDP-glucose binding domain and at least one of eight transmembrane domains. The invention further provides a cellulose synthase promoter, or a functional fragment thereof, which fragment contains one or more mechanical stress response elements (MSRE).

In another aspect, the present invention is directed to a method of improving the quality of wood by altering the quantity of cellulose in plant cells, and optionally decreasing the content of lignin in the cell. The invention also relates to a method of altering the growth or the cellulose content of a plant by expressing an exogenous polynucleotide encoding a cellulose synthase or a UDP-glucose binding domain thereof in the plant. The invention further provides a method for causing a stressinduced gene expression in a plant cell by expressing a polynucleotide of choice using a stress-inducible cellulose synthase promoter.

In yet another aspect, the invention relates to a method for determining a mechanical stress responsive element (MSRE) in a cellulose synthase promoters and a method for identifying transcription factors that binds to the MSRE.

In a further aspect, the invention provides a method for altering (increasing or decreasing) regulating, the expression of a cellulose synthase in a plant by expressing an exogenous polynucleotide encoding a transcription factor having the property of binding a positive MSRE of a cellulose synthase promoter or by expressing an antisense polynucleotide encoding a transcription factor having the property of binding a negative MSRE to block the expression of the transcription factor.

Other aspects of the invention will be appreciated by a consideration of the detailed description of the invention drawings and appended claims.

WO 00/71670 PCT/US00/13637 DESCRIPTION OF THE DRAWINGS Fig. 1 represents a nucleic acid sequence encoding a cellulose synthase from Populus tremuloides [SEQ ID NO: 1] and the protein sequence thereof [SEQ ID NO: 2].

Fig. 2 a-c (collectively referred to as Fig. 2) represent a Southern blot analysis of aspen genomic DNA probed with a fragment of the aspen cDNA represented in Fig. 1 under low (panel a) and high stringency conditions (panel and a northern blot analysis of the total aspen RNA from stem intemodes using the same probe (panel c).

Fig. 3 a-d (collectively referred to as Fig. 3) represent in situ localization of the cellulose synthase gene transcripts as shown in the transverse sections from second (panel fourth (panel sixth (panel c) and fifth (panel d) internode.

Fig. 4 represents a nucleic acid sequence of the 5' region of aspen cellulose synthase gene including the promoter region and the 5' portion of the coding sequence [SEQ ID NO: 3].

Fig. 5 a-f (collectively referred to as Fig. 5) represents a histochemical analysis (panels a-d and f) and fluorescence microscopy (panel e) of transgenic tobacco for GUS gene expression driven by a cellulose synthase promoter of the invention.

Fig. 6 a-d (collectively referred to as Fig. 6) represents a histochemical analysis of GUS gene expression driven by aspen cellulose synthase promoter of the invention; tangential and longitudinal sections were harvested before bending (panel a), and 4 (panel 20 (panel c) and 40 (panel d) hours after bending and stained for GUS expression.

Fig. 7 represents a cDNA encoding cellulose synthase isolated from Arabidopsis [SEQ ID NO:4].

Fig. 8 represents an Arabidopsis cellulose synthase [SEQ ID encoded by the cDNA represented in Fig. 7.

DETAILED DESCRIPTION OF THE INVENTION All patents, patent applications and references cited in this specification are hereby incorporated herein by reference in their entirety. In case of any inconsistency, the present disclosure governs.

Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the person of skill in the art in describing the compositions and methods of the invention and how to make and use them. It will be appreciated that the WO 00/71670 PCTIUSOO/1 3637 -6same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term.

Likewise, the invention is not limited to the preferred embodiments.

The term "plant" includes whole plants and portions of plants, including plant organs roots, stems, leaves, etc.).

The term "angiosperm" refers to plants which produce seeds encased in an ovary. A specific example of an angiosperm is Liquidambar styracifllta (L.)[sweetgum].

The term "gymnosperm" refers to plants which produce naked seeds, that is, seeds which are not encased in an ovary. Specific examples of a gymnosperm include Pinus taeda (L.)[loblolly pine].

The term "polynucleotide" or "nucleic acid molecule" is intended to include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense strands together or individually (although only sense or anti-sense stand may be represented herein). This includes singleand double-stranded molecules, DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thiouracil, thio-guanine and fluoro-uracil.

An "isolated" nucleic acid molecule or polynucleotide refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide may contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. A polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel, for example) from the rest of the cellular components is considered "isolated". The polynucleotides and polypeptides of the invention may be "substantially pure," having the highest degree of purity that can be achieved using purification techniques known in the art.

The term "hybridization" refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. Polynucleotides are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to a strand of another polynucleotide under defined stringency conditions. Hybridization requires that the two polynucleotides contain substantially complementary sequences; WO 00/71670 PCT/USOO/13637 -7depending on the stringency of hybridization, however, mismatches may be tolerated.

Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5X SSC at 65cC) requires that the sequences exhibit some high degree of complementarily over their entire sequence. Conditions of intermediate stringency (such as, for example, an aqueous solution of 2X SSC at 65°C) and low stringency (such as, for example, an aqueous solution of 2X SSC at 55 0 require correspondingly less overall complementarily between the hybridizing sequences. (IX SSC is 0.15 M NaCI, 0.015 M Na citrate.) As used herein, the above solutions and temperatures refer to the probe-washing stage of the hybridization procedure. The term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions" is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide.

A "sequence-conservative variant" is a polynucleotide that contains a change of one or more nucleotides in a given codon position, as compared with another polynucleotide, but the change does not result in any alteration in the amino acid encoded at that position.

A "function-conservative variant" is a polypeptide (or a polynucleotide encoding the polypeptide) having a given amino acid residue that has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). Amino acids with have similar physico-chemical properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Sequence- and function-conservative variants are discussed in greater detail below with respect to degeneracy of the genetic code.

A "functional domain" or a "functional fragment" refers to any region or portion of a protein or polypeptide or polynucleotide which is a region or portion of a larger protein or polynucleotide, the region or portion having the specific activity or specific function attributable to the larger protein or polynucleotide, a functional domain of cellulose synthase is the UDP-glucose binding domain.

The term identity" refers to the percentage of the nucleotides/amino acids of one polynucleotide/polypeptide that are identical to the nucleotides/amino acids of another sequence of polynucleotide/polypeptide as identified by program GAP from Genetics Computer Group Wisconsin (GCG) package (version 9.0) (Madison, WI). GAP uses the algorithm of Needleman and Wunsch Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. When parameters required to run the above algorithm are WO 00/71670 PCT/US00/13637 -8not specified, the default values offered by the program are contemplated. The following parameters are used by the GCG program GAP as default values (for polynucleotides): gap creation penalty:50; gap extension penalty: 3 scoring matrix: nwsgapdna.cpm (local data file).

The similarity" or homology" between two polypeptide sequences is a function of the number of similar positions shared by two sequences on the basis of the scoring matrix used divided by the number of positions compared and then multiplied by 100. This comparison is made when two sequences are aligned (by introducing gaps if needed) to determine maximum homology. PowerBlast program, implemented by the National Center for Biotechnology Information, can be used to compute optimal, gapped alignments. GAP program from Genetics Computer Group Wisconsin package (version (Madison, WI) can also be used. GAP uses the algorithm of Needleman and Wunsch (J Mol Biol 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. When parameters required to run the above algorithm are not specified, the default values offered by the program are contemplated. The following parameters are used by the GCG program GAP as default values (for polypeptides): gap creation penalty:12; gap extension penalty:4; scoring matrix:Blosum62.cpm (local data file).

The term "oligonucleotide" refers to a nucleic acid, generally of at least preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of CelA, or to detect the presence of nucleic acids encoding CelA. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a CelA DNA molecule. In still another embodiment, a library of oligonucleotides arranged on a solid support, such as a silicon wafer or chip, can be used to detect various polymorphisms of interest. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

The term "coding sequence" refers to that portion of the gene that contains the information for encoding a polypeptide. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic WO 00/71670 PCT/USOO/13637 -9sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic mammalian) DNA, and even synthetic DNA sequences.

A "promoter" is a polynucleotide containing elements a TATA box) which are capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.

Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Examples of promoters that can be used in the present invention include PtCelAP, 4CL-1 and The term "constitutive promoter" refers to a promoter which typically, does not require positive regulatory proteins to activate expression of an associated coding sequence, a constitutive promoter maintains some basal level of expression. A constitutive promoter is commonly used in creation of an expression cassette. An example of a constitutive promoter are 35S CaMV (Cauliflower Mosaic Virus), available from Clonetech, Palo Alto, CA.

The term "inducible promoter" refers to the promoter which requires a positive regulation to activate expression of an associated coding sequence. An example of such a promoter is a stress-inducible cellulose synthase promoter from aspen described herein.

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

A "vector" is a recombinant nucleic acid construct, such as plasmid, phage genome, virus genome, cosmid, or artificial chromosome to which a polynucleotide of the invention may be attached. In a specific embodiment, the vector may bring about the replication of the attached segment, in the case of a cloning vector.

The term "expression cassette" refers to a polynucleotide which contains both a promoter and a protein coding sequence such that expression of a given protein is achieved upon insertion of the expression cassette into a cell.

A cell has been "transfected" by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous polynucleotide when the transfected polynucleotide effects a phenotypic change. Preferably, the transforming polynucleotide WO 00/71670 PCT/USOO/13637 should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

"Exogenous" refers to biological material, such as a polynucleotide or protein, that has been isolated from a cell and is then introduced into the same or a different cell. For example, a polynucleotide encoding a cellulose synthase of the invention can be cloned from xylem cells of a particular species of tree, inserted into a plasmid and reintroduced into xylem cells of the same or different species. The species thus contains an exogenous cellulose synthase polynucleotide.

"Heterologous polynucleotide" refers to an exogenous polynucleotide not naturally occurring in the cell into which it is introduced.

"Homologous polynucleotide" refers to an exogenous polynucleotide that naturally exists in the cells into which it is introduced.

The present invention relates to isolation and characterization of polynucleotides encoding cellulose synthases from plants, especially trees, including full length or naturally occurring forms of cellulose synthases, functional domains, promoters and regulatory elements. Therefore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.

See, Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II Glover ed. 1985); Oligonucleotide Synthesis Gait ed. 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. (1985)]; Transcription And Translation Hames S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I.

Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. Current Protocols in Molecular Biology, John Wiley Sons, Inc. (1994).

The present invention relates to a novel, full-length cellulose synthase gene (CelA), a novel stress inducible promoter of cellulose synthases (CelAP), and cellulose synthase proteins from trees, including UDP-glucose catalytic domains thereof. The invention enables the development of transgenic tree varieties having increased cellulose content, decreased lignin content and, therefore, improved wood fiber characteristics.

Production of increased cellulose quantity and quality in multiple varieties of commercially relevant, transgenic forest tree species in operational production scenarios are further contemplated. The invention further provides a new experimental system for study of CelA gene expression and function in trees.

WO 00/71670 PCT/US00/13637 -11- Polynucleotides encoding cellulose synthase and fragments thereof The present invention relates to polynucleotides which comprise the nucleotide sequence that encodes cellulose synthase of the invention or a functional fragment thereof. In a preferred embodiment, the polynucleotide comprises the sequence encoding a tree cellulose synthase and most preferrably, the sequence encoding a cellulose synthase from aspen. In one embodiment, a polynucleotide of the invention includes the entire cellulose synthase coding region, nucleotides 69 to 3,005 of SEQ ID NO: 1. In another aspect of the invention, the polynucleotide encoding an Arabidopsis cellulose synthase is provided (see SEQ ID NO:4 and the translated protein of SEQ ID Also within the scope of the invention are fragments of the polynucleotides encoding cellulose synthase of the invention, which fragments encode at least one transmembrane domain and/or a UDP-glucose binding domain. For example, a polynucleotide comprising the nucleotides encoding a UDP-glucose binding domain of aspen cellulose synthase nucleotides 660 to 2250 of SEQ ID NO:1) or corresponding nucleotides of SEQ ID NO:4 are within the scope of the invention. The nucleotides encoding the UDP-glucose binding domain can be determined by, for example, alignment of protein sequences as described below.

The invention further relates to sequence conservative variants of the coding portion of SEQ ID NOS: 1 and 4.

Polynucleotides that hybridize under conditions of low, medium, and high stringency to SEQ ID NOS: 1 and 4, and their respective coding portions are also within the scope of the invention. Preferably, the polynucleotide that hybridizes to any of SEQ ID NOS: 1 and 4, or their respective coding portions, is about the same length as that sequence, for example, not more than about 10 to about 20 nucleotides longer or shorter.

In another embodiment of the invention, the hybridizable polynucleotide is at least 1500 nucleotides long, preferably at least 2500 nucleotides long and most preferably at least 3000 nucleotides long. In yet another embodiment, the hybridizable polynucleotide comprises the UDP-glucose binding domain as found in SEQ ID NO: 1 or 4, or at least the conserved region QVLRW. Most preferably, the hybridizable polynucleotide has a UDPglucose binding activity.

The polynucleotides that occur originally in nature may be isolated from the organisms that contain them using methods described herein or well known in the art. The non-naturally occurring polynucleotides may be prepared using various manipulations known in the field of recombinant DNA. For example, the cloned CelA polynucleotide can be modified according to methods described by Sambrook et al., 1989. The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the modified polynucleotides, for example, care should be taken to ensure that the modified WO 00/71670 PCT/USOO/13637 -12polynucleotide remains within the appropriate translational reading frame (if to be expressed) or uninterrupted by translational stop signals. As a further example, a CelAencoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Preferably, such mutations enhance the functional activity of the mutated CelA polynucleotide. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984. DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad.

Sci. U.S.A. 83:710), use of TAB linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The polynucleotides of the present invention may be introduced into various vectors adapted for plant or non-plant replication. These are well known in the art, thus, choice, construction and use of such vectors is well within the skill of a person skilled in the art. Possible vectors include, but are not limited to, plasmids or modified viruses of plants, but the vector system must be compatible with the host cell used. An example of a suitable vector is Ti plasmid. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. An expression cassette containing cellulose synthase or recombinant molecules thereof can be introduced into host cells via silicon carbide whiskers, transformed protoplasts, transformation, Agrobacterium vectors (discussed below), electroporation, infection, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid.

Transgenic plants containing the polynucleotides described herein are also within the scope of the invention. Methods for introducing exogenous polynucleotides WO 00/71670 PCT/US00/13637 -13into plant cells and regenerating transgenic plants are well known. Some are provided below.

In one embodiment, to introduce a plasmid containing a CelA coding sequence or promoter of the invention into a plant, a 1:1 mixture of plasmid DNA containing a selectable marker expression cassette and plasmid DNA containing a cellulose synthase expression cassette is precipitated with gold to form microprojectiles.

The microprojectiles are rinsed in absolute ethanol and aliquots are dried onto a suitable macrocarrier such as the macrocarrier available from BioRad in Hercules, CA. Prior to bombardment, embryogenic tissue is preferably desiccated under a sterile laminar-flow hood. The desiccated tissue is transferred to semi-solid proliferation medium. The prepared microprojectiles are accelerated from the macrocarrier into the desiccated target cells using a suitable apparatus such as a BioRad PDS-1000/HE particle gun. In a preferred method, each plate is bombarded once, rotated 180 degrees, and bombarded a second time. Preferred bombardment parameters are 1350 psi rupture disc pressure, 6 mm distance from the rupture disc to macrocarrier (gap distance), 1 cm macrocarrier travel distance, and 10 cm distance from macrocarrier stopping screen to culture plate (microcarrier travel distance). Tissue is then transferred to semi-solid proliferation medium containing a selection agent, such as hygromycin B, for two days after bombardment.

Cellulose synthase protein and fragment thereof A cellulose synthase of the invention is a plant protein that contains a catalytic subunit which has UDP-glucose binding activity for the synthesis of glucan from glucose, and eight transmembrane domains for localizing the cellulose synthase to the cell membrane. The cellulose synthase of the invention has eight transmembrane binding domains; two at the amino terminal and six at the carboxyl terminal. The UDP-glucose binding domain is located between transmembrane domains two and three. Examples of this protein structure are seen in the aspen cellulose synthase as well as in those of RSW1 and GhCelA. The location of the transmembrane domain may be identified as described below and as exemplified in the Example. Preferably, the cellulose synthase of the invention has an amino acid sequence of a tree cellulose synthase.

In one embodiment, the cellulose synthase protein of the invention is isolated from aspen. Aspen cellulose synthase contains about 978 amino acids and has a molecular weight of about 110 KDa and a pi of about 6.58. In one embodiment, the aspen cellulose synthase has the amino acid sequence of SEQ ID NO:2 as represented in Fig. 1.

In another aspect, the invention relates to cellulose synthase of SEQ ID NO: The invention further relates to fragments of plant cellulose synthases, such as fragments containing at least one transmembrane region and/or a UDP-glucose binding WO 00/71670 PCT/USOO/13637 -14domain. The transmembrane regions may be identified as described in the Example by using the method of Hoffman and Stoffel (1993).

The cellulose synthase fragment containing the UDP-glucose binding domain is functional without the presence of the rest of the protein. This separable activity is as shown in the Example. This result was surprising and unexpected because previously identified UDP-glucose binding domains were not known to be functional when isolated from other portions of the protein. Thus, a fragment of any cellulose synthase (such as PtCelA, RSW1, GhCelA and SEQ ID NO:5) that contains a UDP-glucose binding domain and is independently functional is within the scope of the invention. The function of the UDP-glucose binding domain may be determined using the assay described in the Example. The UDP-glucose binding domain of the invention is located between the second and third transmembrane region of the cellulose synthase and has conserved amino acid sequences for UDP-glucose binding, such as the sequence QVLRW and conserved D residues. The UDP-glucose binding domain and the conserved regions therein may be located in a cellulose synthase using the guidance of the present specification and the general knowledge in the art, for example Brown, 1996. In one embodiment, the UDPglucose binding domain and the conserved regions therein may be identified by comparing the amino acid sequence of cellulose synthase of interest with the amino acid sequence of aspen cellulose synthase using the algorithms described in the specification or generally known in the art. For example, the UDP-glucose binding domain of SEQ ID NO:2 is in the position amino acids 220 to 749. The conserved QVLRW sequence is located at positions 715-719 of SEQ ID NO:2.

Polypeptides having at least 75%, preferably at least 85% and most preferably at least 95% similarity to the amino acid sequence of SEQ ID NO: 2, amino acids 220-749 of SEQ ID NO:2, SEQ ID NO:5 or its UDP-glucose binding domain using Power Blast or GAP algorithm described above. In a preferred embodiment, these polypeptides are of about the same length as the polypeptide of SEQ ID NO: 2 or amino acids 220-749 of SEQ ID NO:2. For example, the polypeptide may be from about 2-3 to about 5-7 and to about 10-15 amino acids longer or shorter. In another embodiment, the polypeptides described in this paragraph are not originally found naturally occurring) in Arabidopsis or cotton. These polypeptides may be prepared by, for example, altering the nucleic acid sequence of a cloned polynucleotide encoding the protein of SEQ ID NO:2 or SEQ ID NO:5 using the methods well known in the art.

Function conservative variants of cellulose synthase are also within the scope of the invention and can be prepared by altering the sequence of a cloned polynucleotide encoding cellulose synthase or fragments thereof. Conventional methods used in the art can be used to make substitutions, additions or deletions in one or more amino acids, to provide functionally equivalent molecules. For example, a function WO 00/71670 PCT/US00/13637 conservative variant that has substitutions, deletions and/or additions in the amino and/or carboxyl terminus of the protein, outside of the UDP-glucose binding domain is within the scope of the invention. Preferably, variants are made that have enhanced or increased functional activity relative to native cellulose synthase. Methods of directed evolution can be used for this purpose.

The invention also includes function conservative variants which include altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are: Lys for Arg and vice versa such that a positive charge may be maintained; (ii) Glu for Asp and vice versa such that a negative charge may be maintained; (iii) Ser for Thr such that a free -OH can be maintained; and (iv) Gln for Asn such that a free CONH 2 can be maintained. Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces b-turns in the protein's structure.

The cellulose synthase of the invention can be isolated by expressing a cloned polynucleotide encoding the cellulose synthase as well as using direct protein purification techniques. These methods will be apparent to those of skill in the art.

Polynucleotides containing cellulose synthase promoter The present invention further relates to a cellulose synthase promoter. The promoter is a stress-inducible promoter and may be used to synthesize greater quantities of high crystalline cellulose in plant, and preferably in trees. This permits an increase in the WO 00/71670 PCT/USO/13637 -16proportion of cellulose in transgenic plants, greater strength of juvenile wood and fiber, and acceleration of overall growth rate.

In one embodiment, the promoter of the invention is from aspen and is represented in Figure 4. The promoter sequence is located within the region of nucleotides 1-840 of SEQ ID NO:3. A person of skill in the art will appreciate that not the entire sequence is required for the promoter function and can easily identify the critical regions by looking for conserves boxes and doing routine deletion analysis. Thus, functional fragments of SEQ ID NO: 1 are within the scope of the invention.

Polynucleotides that hybridize under conditions of low, medium, and high stringency to SEQ ID NO:3, and its non-coding portion are also within the scope of the invention. The hybridizable polynucleotide may be about the same length as the sequence to which it hybridizes, for example, not more than about 10 to about 20 nucleotides longer or shorter. In another embodiment, the hybridizable polynucleotide is at least about 200 nucleotides long, preferably at least about 400 nucleotides long and most preferably at least 500 nucleotides long. In yet another embodiment, the hybridizable polynucleotide comprises at least one MSRE element identified according to the method described below.

A cellulose synthase promoter of the invention typically provides tissuespecific gene regulation in xylem, but also permits up-regulation of gene expression in other tissues as well, phloem under tension stress. Furthermore, expression of cellulose synthase is localized to an area of the plant under stress.

This stress-inducible phenomenon is regulated by positive and negative mechanical stress response elements (MSREs). These MSREs upregulate (positive) or downregulate (negative) the expression of a cellulose synthase polynucleotide under stress conditions through binding of transcription factors. MSRE-regulated expression of cellulose synthase permits synthesis of cellulose with high crystallinity.

The MSREs of cellulose synthase can be modified or employed otherwise in methods to regulate expression of a polynucleotide, including a cellulose synthase, operatively linked to a promoter containing an MSRE in response to mechanical stress tension or compression) to a transgenic plant.

Negative MSREs of a cellulose synthase promoter can be modified, removed or blocked to improve expression of a cellulose synthase, and thereby increase cellulose production and improve wood quality. Alternatively, positive MSREs can be removed or blocked to decrease expression of a cellulose synthase, which decreases cellulose production and increases lignin deposition. This is useful for increasing the fuel value of wood because lignin has a higher BTU value than cellulose. Moreover, a modified cellulose synthase promoter can be operatively linked to a polynucleotide of interest to control its expression upon mechanical stress to a plant harboring it.

WO 00/71670 PCT/USO/13637 -17- The location of MSRE elements in the SEQ ID NO:3 may be identified, for example, using promoter deletion analysis, DNAse Foot Print Analysis, and Southwestern screening of an expression library for an MSRE. In one embodiment, cellulose synthase promoter that has one or more portions deleted, and is operatively linked to a reporter sequence, is introduced into a plant or a plant cell. A positive MSRE is detected by observing no relative change or increase in the amount of reporter in a transgenic plant or tissue, phloem after inducing a stress to the plant, and a negative MSRE is detected by observing increases in the amount of reporter in the plant in the absence of any stress to the plant. A positive element is detected when by removing it, GUS expression goes down and by adding it kept at the same level or more. The negative element does not support, or suppreses, expression of GUS and by removing it, normal or enhanced GUS expression is observed as compared to when negative element is present.

Manipulation of a MSRE binding sites and/or providing transcription factors that bind thereto, provides a mechanism to continuously produce high crystalline cellulose in woody plant cell walls of transgenic plants. For example, one having ordinary skill in the art can delete or block negative MSRE elements, or provide cDNA encoding protein(s) that bind the positive MSREs, to enable constitutive expression of a cellulose synthase without the requirement of a mechanical stress. The increased cellulose synthase, and therefore, increased cellulose content, can improve the strength properties of juvenile wood and fiber. It is also contemplated that the positive MSREs can be deleted or blocked, or cDNA in an antisense direction, which in the sense direction encodes a protein that binds a positive MSRE, can be provided, to reduce cellulose synthase activity and decrease cellulose production.

Method of Isolating Polynucleotides Encoding Cellulose Synthase The invention further relates to identifying and isolating polynucleotides encoding cellulose synthase in plants, trees, (in addition to those polynucleotides provided in the Example and represented in Fig. 1 and Fig. These polynucleotides may be used to manipulate expression of cellulose synthase with an objective to improve the cellulose content and properties of wood.

The method comprises identifying a nucleic acid fragment containing a sequence encoding cellulose synthase or a portion thereof by using a fragment of SEQ ID NOS: 1 or 4 as a probe or a primer. Once identified, the nucleic acid fragment containing a sequence encoding cellulose synthase or a portion thereof is isolated.

Polynucleotides encoding cellulose synthases of the invention, whether genomic DNA, cDNA, or fragments thereof, can be isolated from many sources, particularly from cDNA or genomic libraries from plants, preferably trees aspen, sweetgum, loblolly pine, eucalyptus, and other angiosperms and gymnosperms).

WO 00/71670 PCT/US00/13637 -18- Molecular biology methods for obtaining polynucleotides encoding a cellulose synthase are well known in the art, as described above (see, Sambrook et al., 1989, supra).

Accordingly, cells from any species of plant can potentially serve as a nucleic acid source for the molecular cloning of a polynucleotide encoding a cellulose synthase of the invention. The DNA may be obtained by standard procedures known in the art from cloned DNA a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression of a cellulose synthase xylem tissue, since cells in this tissue evidence very high levels of expression of CelA), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from a desired cell (see, for example, Sambrook et al., 1989, supra; Glover, D.M. 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, a polynucleotide should be molecularly cloned into a suitable vector for its propagation.

In another embodiment for the molecular cloning of a polynucleotide encoding a cellulose synthase of the invention from genomic DNA, DNA fragments are generated from a genome of interest, such as from a plant, or more particularly a tree genome, part of which will correspond to a desired polynucleotide. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing a desired CelA sequence may be accomplished in a number of ways.

For example, if an amount of a portion of a CelA sequence or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated

DNA

fragments may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci.

U.S.A. 72:3961). For example, a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for a CelA protein from trees can be prepared and used as probes for DNA encoding cellulose synthase, or as primers for cDNA or mRNA in combination with a poly-T primer for RT-PCR). Preferably, a fragment is selected that is highly unique to a cellulose synthase of the invention, such as the UDPglucose binding regions. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent WO 00/71670 PCT/US00/13637 -19hybridization conditions can be used. In a specific embodiment, stringency hybridization conditions can be used to identify homologous CelA sequences from trees or other plants.

Thus, in one embodiment, a labeled cellulose synthase cDNA from, e.g., Populus tremuloides (PtCelA), can be used to probe a library of genes or DNA fragments from various species of plants, especially angiosperm and gymnosperm, to determine whether any bind to a CelA of the invention. Once genes or fragments are identified, they can be amplified using standard PCR techniques, cloned into a vector, pBluescript vector (StrataGene of LaJolla, CA), and transformed into a bacteria, DH5a E. coli strain (Gibco BRL of Gaithersburg, MD). Bacterial colonies are typically tested to determine whether any contains a cellulose synthase-encoding nucleic acid. Once a positive clone is identified through binding, it is sequenced from an end, preferably the 3' end.

cDNA libraries can be constructed in various hosts, such as lambda ZAPII, available from Stratagene, LaJolla, CA, using poly(A) +RNA isolated from aspen xylem, according to the methods described by Bugos et al. (Biotechniques 19:734-737, 1995 The above mentioned probes are used to assay the aspen cDNA library to locate cDNA which codes for enzymes involved in production of cellulose synthases. Once a cellulose synthase sequence is located, it is then cloned and sequenced according to known methods in the art.

Further selection can be carried out on the basis of the properties of the gene, if the gene encodes a protein product having the isoelectric, electrophoretic, hydropathy plot, amino acid composition, or partial amino acid sequence of a cellulose synthase protein of the invention, as described herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones or DNA clones which hybrid-select the proper mRNAs can be used to produce a protein that has similar properties known for cellulose synthases of the invention. Such properties may include, for example, similar or identical electrophoretic migration patterns, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, hydropathy plots, or functional properties (such as isolated, functional UDP-glucose binding domains).

A cellulose synthase polynucleotide of the invention can also be identified by mRNA selection, by nucleic acid hybridization followed by in vitro translation. In this procedure, nucleotide fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified CelA DNA, or may be synthetic oligonucleotides designed from the partial amino acid sequence information.

Functional assays UDP-glucose activity) of the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments, that contain the desired sequences.

WO 00/71670 PCTfUSOO/13637 A radiolabeled CelA cDNA can be synthesized using a selected mRNA as a template. The radiolabeled mRNA or cDNA may then be used as a probe to identify homologous CelA DNA fragments from amongst other genomic DNA fragments.

It will be appreciated that other polynucleotides, in addition to a CelA of the invention can be operatively linked to a CelA promoter to control expression of the polynucleotide upon application of a mechanical stress.

Expression of CelA Polypeptides The nucleotide sequence coding for CelA, or a functional fragment, derivative or analog thereof, including chimeric proteins, can be inserted into an appropriate expression vector, a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Preferably, an expression vector includes an origin of replication. The elements are collectively termed herein a "promoter." Thus, a nucleic acid encoding CelA of the invention can be operatively associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding CelA and/or its flanking regions.

In addition to a CelAP, expression of cellulose synthase can be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control CelA polynucleotide expression include, constitutive, development-specific and tissuespecific. Examples of these promoters include 35S Cauliflower Mosaic Virus, terminal flower and 4CL-1. Thus, there are various ways to alter the growth of a plant using different promoters, depending on the needs of the practitioner.

The nucleotide sequence may be inserted in a sense or antisense direction depending on the needs of the practitioner. For example, if augmentation of cellulose biosynthesis is desired then polynucleotides encoding, cellulose synthase, can be inserted into the expression vector in the sense direction to increase cellulose synthase production and thus cellulose biosynthesis. Alternatively, if it is desired that cellulose biosynthesis is reduced or lignin content is increased, then polynucleotides encoding, e.g., cellulose synthase ,can be inserted in the antisense direction so that upon transcription the antisense mRNA hybridizes to other complementary transcripts in the sense orientation to prevent translation. In other embodiments, the polynucleotide encodes a UDP-glucose binding domain and is used in a similar manner as described.

A recombinant CelA protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after WO 00/71670 PCT/US00/13637 -21integration of the coding sequence by recombination. In this regard, any of a number of amplification systems for plants may be used to achieve high levels of stable gene expression, as discussed above. Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).

Expression vectors containing a nucleic acid encoding a CelA of the invention can be identified by four general approaches: PCR amplification of the desired plasmid DNA or specific mRNA, nucleic acid hybridization, presence or absence of selection marker gene functions, analyses with appropriate restriction endonucleases, and expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions 3-glucuronidase activity, resistance to antibiotics, transformation phenotype. etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding CelA is inserted within the "selection marker" gene sequence of the vector, recombinants containing the CelA insert can be identified by the absence of the CelA gene function. In the fourth approach, recombinant expression vectors are identified by digestion with appropriate restriction enzymes. In the fifth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.

After a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to those vectors or their derivatives described above.

Vectors are introduced into the desired host cells by methods known in the art, Agrobacterium-mediated transformation (described in greater detail below), transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and WO 00/71670 PCT/US00/13637 -22- Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

The cell into which the recombinant vector comprising the nucleic acid encoding CelA is cultured in an appropriate cell culture medium under conditions that provide for expression of CelA by the cell. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (such as glycosylation, cleavage, of a signal sequence) of proteins.

Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

Agrobacterium-mediated transformation and inducing somatic embryos The culture media used in the invention, and for transforming Agrobacterium, contain an effective amount of each of the medium components basal medium, growth regulator, carbon source) described above. As used in describing the present invention, an "effective amount" of a given medium component is the amount necessary to cause a recited effect. For example, an effective amount of a growth hormone in the primary callus growth medium is the amount of the growth hormone that induces callus formation when combined with other medium components. Other compounds known to be useful for tissue culture media, such as vitamins and gelling agents, may also be used as optional components of the culture media of the invention.

Transformation of cells from plants, trees, and the subsequent production of transgenic plants using Agrobacterium-mediated transformation procedures known in the art, and further described herein, is one example of a method for introducing a foreign gene into trees. Transgenic plants may be produced by various methods, such as by the following steps: culturing Agrobacterium in low-pH induction medium at low temperature and preconditioning, coculturing bacteria with wounded tobacco leaf extract in order to induce a high level of expression of the Agrobacterium vir genes whose products are involved in the T-DNA transfer; (ii) coculturing a desired plant tissue explants, including zygotic and/or somatic embryo tissues derived from cultured explants, with the incited Agrobacterium; (iii) selecting transformed callus tissue on a medium containing antibiotics; and and converting the embryos into plantlets.

Any non-tumorigenic A. tumefaciens strain harboring a disarmed Ti plasmid may be used in the method of the invention. Any Agrobacterium system may be used. For example, Ti plasmid/binary vector system or a cointegrative vector system with one Ti plasmid may be used. Also, any marker gene or polynucleotide conferring the ability to select transformed cells, callus, embryos or plants and any other gene, such as, WO 00/71670 PCT/US00/13637 -23for example ,a gene conferring resistance to a disease, or one improving cellulose content, may also be used. Any promoter desired can be used, such as, for example, a PtCelAP of the invention, and those promoters as described above. A person of ordinary skill in the art can determine which markers and genes are used depending on particular needs.

For purposes of the present invention, "transformed" or "transgenic" means that at least one marker gene or polynucleotide conferring selectable marker properties is introduced into the DNA of a plant cell, callus, embryo or plant. Additionally, any gene may also be introduced.

To increase the infectivity of the bacteria, Agrobacterium is cultured in low-pH induction medium, any bacterium culture media with a pH value adjusted to from 4.5 to 6.0, most preferably about 5.2, and at low temperature such as for example about 19-30 0 C, preferably about 21-26 0 C. The conditions of low-pH and low temperature are among the well-defined critical factors for inducing virulence activity in Agrobacterium Altmorbe et al., Mol. Plant-Microbe. Interac. 2: 301, 1989; Fullner et al., Science 273: 1107, 1996; Fullner and Nester, J. Bacteriol. 178: 1498, 1996).

The bacteria is preconditioned by coculturing with wounded tobacco leaf extract (prepared according to methods known generally known in the art) to induce a high level of expression of the Agrobacterium vir genes. Prior to inoculation of plant somatic embryos, Agrobacterium cells can be treated with a tobacco extract prepared from wounded leaf tissues of tobacco plants grown in vitro. To achieve optimal stimulation of the expression of Agrobacterium vir genes by wound-induced metabolites and other cellular factors, tobacco leaves can be wounded and pre-cultured overnight. Culturing of bacteria in low pH medium and at low temperature can be used to further enhance the bacteria vir gene expression and infectivity. Preconditioning with tobacco extract and the vir genes involved in the T-DNA transfer process are generally known in the art.

Agrobacterium treated as described above is then cocultured with a plant tissue explant, such as for example zygotic and/or somatic embryo tissue. Non-zygotic somatic) or zygotic tissues can be used. Any plant tissue may be used as a source of explants. For example, cotyledons from seeds, young leaf tissue, root tissues, parts of stems including nodal explants, and tissues from primary somatic embryos such as the root axis may be used. Generally, young tissues are a preferred source of explants.

The invention also relates to methods of altering the growth of a plant by expressing the polynucleotide of the invention, which as a result alters the growth of the plant. The polynucleotide used in the method may be a homologous polynucleotide or a heterologous polynucleotide and are described in detail above. For example, both fulllength and UDP-glucose binding region containing fragments may be expressed.

Additionally, depending on the aim of the method, the polynucleotide may be introduced into the plant in the sense or in the antisense orientation. Any suitable promoter may be WO 00/71670 PCT/US00/13637 -24used to provide expression. The promoter or a functional fragment thereof is operatively linked to the polynucleotide. The promoter may be a constitutive promoter, a tissuespecific promoter or a development-specific plant promoter. Examples of suitable promoters are Cauliflower Mosaic Virus 35S, 4CL, cellulose synthase promoter, PtCelAP and terminal flower promoter.

The invention further relates to a method of altering the cellulose content in a plant by expressing the polynucleotide of the invention as described above. The method may be used to increased the ratio of cellulose to lignin in the plant that have an exogenous polynucleotide of the invention introduced therein.

The invention further relates to a method for altering expression of a cellulose synthase in a plant cell by introducing into the cell a vector comprising a polynucleotide of the invention and expressing the polynucleotide. The polynucleotides and promoters described above may be used.

A method for causing stress-induced gene expression in a plant cell is also within the scope of the invention. The method comprises introducing into the plant or a plant cell an expression cassette comprising a cellulose synthase promoter or a functional fragment thereof or providing a plant or a plant cell that comprises the expression cassette (The promoter of the cassette is operatively linked to a coding sequence of choice.); and (ii) applying mechanical stress to the plant to induce expression of the desired coding sequence.

A method for determining a positive mechanical stress responsive element (MSRE) in a cellulose synthase promoter is also within the scope of the invention and comprises making serial deletions in the cellulose synthase promoter, such as for example, SEQ ID NO:3; (ii) introducing the deletion linked to a polynucleotide encoding a reporter sequence into a plant cell, and (iii) detecting a decrease in the amount of reporter in the plant after inducing a stress to the plant. Similarly, a method for determining a negative MSRE in a cellulose synthase promoter is provided. It comprises making serial deletions in the cellulose synthase promoter, such as for example, SEQ ID NO:3; (ii) introducing the deletion linked to a polynucleotide encoding a reporter sequence into a plant cell, and (iii) detecting an increase in the amount of reporter in the plant after inducing a stress to the plant.

The following methods are also within the scope of the invention: a method for expressing cellulose synthase in a tissue-specific manner comprising transforming a plant with a tissue specific promoter operatively linked to a polynucleotide encoding a cellulose synthase; a method for inducing expression of a cellulose synthase in a plant comprising introducing into a plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter, thereby resulting in increased expression of cellulose in the plant, wherein the binding to the positive MSRE results in expression of WO 00/71670 PCT/USOO0/13637 a cellulose synthase; a method for reducing expression of a cellulose synthase comprising introducing into a plant a cDNA in an antisense orientation, wherein the cDNA in a sense orientation encodes a protein that binds to a positive MSRE and results in expression of a cellulose synthase; a method for increasing cellulose biosynthesis in a plant comprising introducing into a plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter, whereby binding of the protein to the positive MSRE results in expression of a cellulose synthase, and A method for reducing cellulose biosynthesis in a plant comprising introducing into a plant a cDNA in an antisense orientation, wherein the cDNA in a sense orientation encodes a protein that binds to a positive MSRE of a cellulose synthase promoter.

EXAMPLE

Molecular cloning of cellulose synthase This Example describes the first tree cellulose synthase cDNA (PtCelA, GenBank No. AF072131) cloned from developing secondary xylem of aspen trees using RSW1 cDNA.

Prior to the present invention, only partial clones of cellulose synthases from crop species and cotton GhCelA have been discovered, which have significant homology to each other. The present inventors have discovered and cloned a new fulllength cellulose synthase cDNA, AraxCelA (GenBank No. AF062485) (Fig. 7, [SEQ ID NO: from an Arabidopsis primary library. AraxCelA is a new member of cellulose synthase and shows 63-85% identity and 72-90% similarity in amino acid sequence with other Arabidopsis CelA members.

Another cellulose synthase was cloned in aspen using a 2 P-labeled 1651-bp long EcoRI fragment of Arabidopsis CelA cDNA, which encodes a centrally located UDPglucose binding domain, was used as a probe to screen about 500,000 pfu of a developing xylem cDNA library from aspen (Populus tremuloides) (Ge and Chiang, 1996). Four positive clones were obtained after three rounds of plaque purification. Sequencing the 3' ends of these four cDNAs showed that they were identical clones. The longest cDNA clone was fully sequenced and determined to be a full-length cDNA having a 3232 bp nucleotide sequence (Fig. 1) [SEQ ID NO: which encodes a protein of 978 amino acids [SEQ ID NO: 2].

Characterization of a cellulose synthase from aspen The first AUG codon of PtCelA was in the optimum context for initiation of transcription on the basis of optimal context sequence described by Joshi (1987a) and Joshi et al. (1997). A putative polyadenylation signal (AATACA) was found 16 bp upstream of a polyadenylated tail of 28 bp, which is similar to the proposed plant structure (Joshi, 1987b). The 5' untranslated leader was determined to have 68 bp and the 3' WO 00/71670 PCTIUSOO/13637 -26untranslated trailor was 227 bp. Both of these regions have a typical length observed in many plant genes (Joshi, 1987a and Joshi, 1987b). This cDNA clone exhibited amino acid sequence similarity with cellulose synthase from cotton (GhCelA,) and 71% with cellulose synthase from Arabidopsis (RSW1), suggesting that this particular tree homolog also encodes a cellulose synthase.

The full length cDNA was designated PtCelA, and encodes a 110,278 Da polypeptide having an isoelectric point (pI) of 6.58 and 8 charged molecules. The hydropathy curve indicated that this particular cellulose synthase has eight transmembrane binding domains; two at the amino terminal and six at the carboxyl terminal, using the method of Hoffman and Stoffel (1993). This protein structure is analogous to those of RSW1 and GhCelA. All of the conserved domains for UDP-glucose binding, such as QVLRW and conserved D residues, are also present in a cellulose synthase of the invention, PtCelA (Brown et al., 1996). Thus, based on sequence and molecular analyses, it was concluded that PtCelA encodes a catalytic subunit which, like RSW1 in Arabidopsis, is essential for the cellulose biosynthesis machinery in aspen.

In situ localization of PtCelA mRNA transcripts along the developmental gradient defined by stem primary and secondary growth demonstrated that cellulose synthase expression is confined exclusively to developing xylem cells undergoing secondary wall thickening. This cell-type-specific nature of PtCelA gene expression was also consistent with xylem-specific activity of cellulose synthase promoter (PtCelAP) based on heterologous promoter-B-glucuronidase (GUS) fusion analysis. Overall, the results provide several lines of evidence that cellulose synthase is the gene primarily responsible for cellulose biosynthesis during secondary wall formation in woody xylem of trees, such as aspen. Previous results by the inventors (Hu et al., 1999) showed that cellulose and lignin are deposited in a compensatory fashion in wood. The discovery of a cellulose synthase in trees, such as aspen, permits the up-regulation of the protein to elevate cellulose production. Surprisingly, expression of CelA in trees suppressed lignin biosynthesis to further improve wood properties of trees.

Preparation of transgenic plants The UDP-glucose binding sequence was subcloned into pBI121, which was used to prepare transgenic tobacco plants (Hu et al., 1998). The expression of a heterologous UDP-glucose binding sequence resulted in a remarkable growth-accelerating effect. This was surprising because current knowledge of the function of plant cellulose synthases teaches that a UDP-glucose sequence must remain intact with other functional domains in CelA, the transmembrane domains, in order for cellulose synthase to initiate cellulose biosynthesis. The remarkable growth and tremendous increase in plant biomass observed in transgenic tobacco was due likely to an augmented deposition of WO 00/71670 PCT/US00/13637 -27cellulose, indicating that the UDP-glucose domain alone is sufficient for genetic augmentation of cellulose biosynthesis in plants.

Genome organization and expression of a novel cellulose svnthase To confirm that the cDNA clone of Fig. 1 [SEQ ID NO: 1] was a cellulose synthase, genomic Southern blot analysis was performed under both high and low stringency conditions using the cDNA. Genomic DNA from aspen was digested with PstI (lane HindII (lane H) and EcoRI (lane and probed using a 1kb 32 P-labeled fragment from the 5' end of a cellulose synthase of Fig. 1. The Southern blot suggested the presence of a small family of cellulose synthase genes in aspen genome (Fig. 2, panels a and Repeated screening of the aspen xylem cDNA library with various plant CelA gene-related probes always resulted in the isolation of the same cellulose synthase cDNA clone. This suggested that the cellulose synthase cDNA cloned (Fig. 1) [SEQ ID NO: 1], represents the primary and most abundant cellulose synthase-encoding gene in developing xylem of trees, such as aspen, where active cellulose deposition takes place. It also indicates that manipulation of cellulose synthase gene expression can have a profound influence on cellulose biosynthesis in trees.

In situ hybridization Northern blot analysis of total RNA from the internodes of aspen seedling stems (Fig. 2, panel c) using the labeled probe (as described above) revealed the near absence of cellulose synthase transcripts in tissues undergoing primary growth (internodes 1 to and that the presence of cellulose synthase transcripts occurs during the secondary growth of stem tissues (intemodes 5 to 11). However, weak northern signals in primary growth may only suggest that cellulose synthase gene expression is specific to xylem, of which there is little in primary growth tissue.

Xylogenesis in higher plants offers a unique model that involves sequential execution of cambium cell division, commitment to xylem cell differentiation, and culmination in xylem cell death (Fukuda, 1996). Although primary and secondary xylem cells originate from different types of cambia, namely procambium and inter/intrafasicular cambium, both exhibit conspicuous secondary wall development with massive cellulose and lignin deposition (Esau, 1965). To further investigate spatial and temporal cellulose synthase gene expression patterns at the cellular level, in situ hybridization was used to localize cellulose synthase mRNA along the developmental gradient defined by stem primary and secondary growth.

Localization of cellulose synthase gene transcripts (RNA) in stem at various growth stages was also observed. Fig. 3 shows transverse sections from 2 nd 4th and 6 th internodes hybridized with digoxygenin (DIG)-labeled cellulose synthase antisense or sense (control) RNA probes, as described.

WO 00/71670 PCT/US00/13637 -28- PtCelA transcripts were detected in young aspen stem sections by in situ hybridization with transcripts of highly variable 5' region of PtCelA cDNA (a 771 bp long fragment generated from PstI and SacI). This region was first subcloned in the plasmid vector, pGEM,-3Zf (Promega) for the production of digoxygenin (DIG)-labeled transcripts using T7 (for antisense transcripts) and SP6 (for sense transcripts) RNA polymerase (DIG system: Boehringer Mannheim). Probes were subjected to mild alkaline hydrolysis by incubation in 100 mM NaHCO 3 pH 10.2 at 60 which produced approximately 200 bp fragments.

Aspen young stems were prepared for sectioning by fixation in 4% (w/v) paraformaldehyde in 100 mM phosphate buffer (pH 7.0) at 4 °C overnight, dehydrated through an ethanol series on ice, and embedded in Paraplast medium (Sigma). Ten pm sections were mounted on Superfrost/plus (Fisher) slides at 42 °C overnight, dewaxed and then rehydrated through a descending ethanol series. The sections were incubated with proteinase K (10 Ag/ml in 100 mM Tris-HC1, 50 mM EDTA, pH 7.5) for 30 min and were post-fixed with FAA. The sections were acetylated with 0.33% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) prior to hybridization. The sections were then incubated in a hybridization mixture (approximately 2 tg/ml DIG-labeled probes, formamide, 2 X SSPE, 10% dextran sulfate, 125 pg/ml tRNA, pH 7.5) at 45 °C for 12-16 hrs. Nonhybridized single-stranded RNA probe was removed by treatment with 20 pg/ml RNase A in TE buffer with 500 mM NaCI. The sections were washed at 50 °C.

Hybridized DIG-labelled probe was detected on sections using anti-digoxygenin antiserum at a 1:1500 dilution, as described in the manufacturer's instruction (DIG system: Boehringer Mannheim). Sections were examined by Eclipse 400 light microscope (Nikon) and photographed.

During the primary growth stage (Fig. 3, panels a and strong expression of cellulose synthase was found localized exclusively to primary xylem (PX) cells. At this stage, young internodes are elongating, resulting in thickening of primary xylem cells through formation of secondary walls (Esau, 1968). The concurrence of shoot elongation with high expression of cellulose synthase strongly suggests the association of cellulose synthase protein with secondary cell wall cellulose synthesis. Later stages of primary growth (Fig. 3, panel b) are characterized by the appearance of an orderly alignment of primary xylem cells. Active cellulose biosynthesis accompanies cell elongation-induced wall thickening, as indicated by the strong expression of cellulose synthase in these primary xylem cells.

At the beginning of secondary growth in older internodes, it was observed that expression of cellulose synthase is also exclusively localized to xylem cells (Fig. 3, panel Instead of elongation in internodes distal to the meristematic activity, growth at this stage is mainly radial due to thickening in secondary cell walls of secondary xylem.

WO 00/71670 PCT/USOO0/13637 -29- At the same time, expression of PtCelA gene becomes localized to the secondary developing xylem cells (SX in Fig. 3, panel which is again consistent with the idea that PtCelA encodes a secondary cell wall cellulose synthase. At this stage, secondary xylem cells cover the elongated and differentiated primary xylem cells in which PtCelA gene expression is no longer detectable (Fig. 3, panel These results demonstrate that expression of PtCelA gene is xylem-specific and the cellulose synthase of Fig. 1 [SEQ ID NO: 1] encodes a cellulose synthase associated with cellulose biosynthesis in secondary walls of xylem cells. To further confirm xylem-specific expression of cellulose synthase, a cellulose synthase gene promoter sequence was cloned and characterized for regulatory activities.

Characterization of expression regulated by cellulose synthase promoter A 5' 1,200 bp cDNA fragment of a cellulose synthase of Fig. 1 [SEQ ID NO: 1] was used as a probe to screen an aspen genomic library for 5' regulatory sequences of a novel cellulose synthase gene, PtCelA. The library was constructed by cloning aspen genomic DNA fragments, generated from an Sau3AI partial-digest and sucrose gradientselected, into the BamHI site of a Lambda DASH II vector (Stratagene, La Jolla, CA).

Five positive clones were obtained from about 150,000 pfu and Lambda DNA was purified. One clone having about a 20 kb DNA insert size was selected for restriction mapping and partial sequencing. This resulted in the identification of a 5' flanking region of PtCelA gene of approximately 1 kb. This genomic fragment, designated PtCelAP (Fig.

4) [SEQ ID NO: contained about 800 bp of promoter sequence, 68 bp of 5' end untranslated region and 160 bp of coding sequence. To investigate regulation of tissuespecific cellulose synthase expression at the cellular level, promoter activity was analyzed in transgenic tobacco plants by histochemical staining of a GUS protein. A PtCelAP-GUS fusion binary vector was constructed in pBI121 with the 35S promoter replaced with PtCelAP [SEQ ID NO: 3] and introduced into tobacco (Nicotiana tabacum) as per Hu et al. (1998).

Eleven independent transgenic lines harboring a CelAP-GUS fusion were generated. Fig. 5 shows a histochemical analysis of GUS expression driven by a cellulose synthase promoter of the invention in transgenic tobacco plants. Transverse sections from the 3rd (panel 5th (panel 7th (panel and 8th (panels d and f) intemodes were stained from GUS activity, and fluorescence microscopy was used to visualize expression under UV radiation.

GUS staining was detected exclusively in xylem tissue of stems, roots and petioles. In stems, strong GUS activity was found localized to xylem cells undergoing primary (Fig. 5, panel a) and secondary growth (Fig. 5 panels b-d and GUS expression was confined to xylem cells in the primary growth stage and became more localized in developing secondary xylem cells during secondary growth. An entire section from the WO 00/71670 PCT/US00/!3637 8th internode stained for GUS activity (Fig. 5, panel These results are consistent with the in vivo expression patterns of cellulose synthase in aspen stems. Lignin autofluorescence was visualized after UV radiation. Phloem fibers, which are also active in cellulose and lignin biosynthesis (Fig. 5, panels d and did not show GUS activity, suggesting that cellulose synthase gene expression is not associated with cellulose biosynthesis in cell types other than xylem. Examination of GUS activity in roots, stems, leaves, anthers and fruit also showed GUS expression in xylem tissue of all these organs suggesting that cellulose synthases of the invention are xylem-specific cellulose and expressed in all plant organs.

Characterization of promoter activity and cellular expression of a cellulose synthase of the invention from one particular source (aspen) indicated hat expression produces a protein that encodes a secondary cell wall-specific cellulose synthase and is specifically compartmentalized in developing xylem cells. Characterization of the cellulose synthase gene promoter sequence not only confirms cell type-specific expression of cellulose synthase, but also provides a method for over-expressing cellulose synthase in a tissue-specific manner to augment cellulose production in xylem.

Expression of cellulose synthase under tension stress As described earlier, a cellulose synthase promoter of the invention is involved in a novel gene regulatory phenomenon of cellulose synthase. To further characterize a cellulose synthase of the invention, GUS expression driven by an aspen cellulose synthase promoter (PtCelAP) was observed in transgenic tobacco plants without or under tension stress. The stress was induced by bending and affixing the plants to maintain the bent position tying) over a 40 hour period. Tangential and longitudinal sections were taken before bending, and 4 hrs, 20 hrs and 40 hrs after bending (panels a-d, respectively).

The cellulose synthase promoter-GUS fusion binary constructs showed exclusive xylem-specific expression of GUS without any tension stress (Fig. 6, panel a).

However, under tension stress conditions endured by angiosperms in nature, the transgenic tobacco plants induced xylem and phloem-specific expression on the upper side of the stem within the first four hours of stress (Fig. 6, panel b).

This observation was surprising because during tension wood development fibers produce highly crystalline cellulose in order to provide essential mechanical strength to a bending stem. The present observation was the first showing of transcriptional upregulation of a cellulose synthase, mediated through a cellulose synthase promoter that is directly responsible for development of highly crystalline cellulose in trees. Furthermore, after 20 hrs of tension stress, both xylem and phloem exhibited GUS expression, but only on the upper side of the stem that was under tensile stress, GUS expression on the lower side was inhibited (Fig. 6, panel With extended stress (up to 40 hrs), GUS WO 00/71670 PCT/US00/13637 -31expression was restricted to only one small region on the upper side of the stem where maximum tension stress was present (Fig. 6, panel Based on the observation of GUS signal in woody cells upon tension stress and the absence of GUS under compression or no stress, it was concluded that a cellulose synthase promoter of the invention has mechanical stress responsive elements (MSREs) that turn cellulose synthase genes on and off depending on the presence and type of stress to the stem.

The results indicate that positive MSREs exist in a cellulose synthase promoter of the invention to bind transcription factors in response to tension stress for regulating the expression of cellulose synthase and increasing biosynthesis of higher crystalline cellulose. This is evident based on the expression of GUS in xylem and phloem tissue at the upper side of the stem subjected to tension stress, but not when tissue on the lower side was subjected to compression or no stress. Furthermore, the tissue at the lower side of the stem, which was subjected to compression stress, showed no GUS expression, expression was turned off. This indicated the presence of negative MSREs, which bind transcription factors to turn off expression of cellulose synthase at the lower side of the stem. Negative MSREs likely suppress development of highly crystalline cellulose in normal wood.

These results proyide a mechanism for genetically engineering synthesis of highly crystalline cellulose in juvenile wood for enhancing strength properties, and for synthesizing a higher percentage of cellulose in reaction wood. The positive MSREs and their cognate transcription factors are important in the synthesis of highly crystalline cellulose of high tensile strength, as are the negative MSREs and inhibition of cognate transcription factors thereto. The present invention thus provides a starting point for cloning cDNAs for the transcription factors that bind to positive and negative MSREs according to methods known in the art. Constitutive expression of cDNAs for positive MSRE transcription factors allows the continuous production of highly crystalline cellulose in transgenic trees, while expression of antisense cDNAs for negative MSRE transcription factors inhibits those transcription factors so that cellulose synthase cannot turn off. This combination will assure continuous production of highly crystalline cellulose in trees.

Genetic engineering of cellulose synthase in transgenic plants As discussed above, the nucleotide sequence of a cellulose synthase of the invention, PtCelA cDNA from aspen, shows significant homology with other polynucleotides encoding cellulose synthase proteins that have been suggested as authentic cellulose synthase clones. To further characterize the activity of a cellulose synthase, four constructs were prepared in a PBI121 plasmid.

1) A constitutive plant promoter Cauliflower mosaic Virus 35S was operatively linked to PtCelA (35SP-PtCelA-s) and overexpressed in transgenic plants.

WO 00/71670 PCT/USOO/13637 -32- This causes excess production of cellulose, resulting in a reduction in lignin content.

Tobacco and aspen have been transformed with this construct.

2) Cauliflower mosaic Virus 35S was operatively linked to antisense RNA from PtCelA (35S-PtCelA-a) and constitutively expressed to reduce production of cellulose and increase lignin content in transgenic plants. This negative control construct may not result in healthy plants since cellulose is essential for plant growth and development. Aspen plants have been transformed with this construct.

3) Aspen 4CL-1 promoter (Hu et al., 1998) was operatively linked to PtCelA (Pt4CLP-PtCelA) (the 35S promoter of PBI121 was removed in this construct) and expressed in a tissue-specific manner in developing secondary xylem of transgenic aspen.

This expression augments the native cellulose production and reduces lignin content of angiosperm tissues. Tobacco and aspen have transformed with this construct.

4) The cytoplasmic domain of PtCelA which contains three conserved regions thought to be involved in UDP-glucose binding during cellulose biosynthesis, was linked to a 35S promoter to produce binary constructs (35S-PtCelA UDP-glucose).

Expression by this promoter permits constitutive expression of a UDP glucose binding domain of PtCelA in transgenic plants. Tobacco and aspen have been transformed with this construct.

constructs (pBI121, ClonTech, CA) were used as controls for each experiment with the constructs. Transgenic tobacco plants were transformed with the constructs. The following table shows the general growth measurements of the TO tobacco plants. Plants carrying a PtCelA construct grew much faster than control plants carrying a pBI121 (control) construct. In comparing developmental 4CL and constitutive promoter control of PtCelA expression, the 35S was more effective, permitting faster growth of transgenic tobacco plants. The fastest growth was seen in transgenic plants carrying a 35S promoter driven UDP-G domain from PtCelA.

It is noted that TO generation plants can have carry over effects from their tissue culture treatments. Therefore, seeds were collected for testing this growth phenomenon in TI generations. The transgenic tobacco plants were analyzed for presence of the transferred genes and all tested positive for the respective gene constructs.

TABLE

Transgenic tobacco plant measurements after transfer in soil for about 1.5 months (N=2) Construct Height Diameter Internode length No. of leaves Longest leaf 17 0.5 11 17 77 1.0 6 13 37 83 1.0 6 13 37 4CLP-PtCelA 41 0.8 5 10 29 Note: All values were measured in centimeters, excluding number of leaves.

It will be appreciated by persons of ordinary skill in the art that the examples and preferred embodiments herein are illustrative, and that the invention may be practiced in a *variety of emodiments which share the same inventive concept.

The word 'comprising' and forms of the word 'comprising' as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions.

*o *•o 7718456.01 WO 00/71670 PCT/US00/13637 -34-

BIBLIOGRAPHY

Hu et al., 1999, Nature Biotechnology, In Press Whetten et al., 1998, Ann Rev PI Physiol P1 Mol Biol, 49: 585-609 Arioli et al., 1998, Science, 279: 717-720 Wu et al., 1998, PI Physiol, 117: 1125 Hu et al., 1998, PNAS, 95: 5407-5412 Joshi et al., 1997, PMB, 35: 993-1001 Fukuda, 1996, Ann Rev P1 Physiol PI Mol Biol, 47: 299-325 Pear et al., 1996, PNAS, 93: 12637-12642 Haigler and Blanton, 1996, PNAS, 93: 12082-12085 Ge and Chiang, 1996, PI Physiol, 112: 861 Brown et al., 1996, Trends P1 Sci., 1: 149-156 Delmer and Amor, 1995, PI Cell, 7: 987-1000 Hoffman and Stoffel, 1993, Biol Chem, Hoppe-Seyler 374: 166 Joshi, 1987, NAR, 15: 6643-6653 Joshi, 1987, NAR, 15: 9627-9640 Timmell, 1986, Compression Wood in Gymnopserms, Springer Verlag Esau, 1967, Plant Anatomy, Wiley and sons, NY Higuchi, 1997, Biochemistry and Molecular Biology of Wood, Springer Verlag EDITORIAL NOTE APPLICATION NUMBER 50262/00 The following Sequence Listing pages 1 to 15 are part of the description. The claims pages follow on pages "35" to "38".

WO 00/71670 PCT/US00/13637 SEQUENCE LISTING <110> Board of Control of Michigan Technological Univers <120> METHOD FOR ENHANCING CELLULOSE AND MODIFYING LIGNIN BIOSYNTHESIS IN PLANTS <130> 66040/9675 <140> <141> <150> 60/135,280 <151> 1999-05-21 <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 3232 <212> DNA <213> Populus tremuloides <220> <221> CDS <222> (69)..(3002) <400> 1 gtcgacccac gcgtccgtct tgaaagaata taagcaag atg atg gaa tct ggg gct Met Met Glu Ser Gly Ala 1 5 tgaagttgta aagagctggt aaagtggtaa cct ata tgc cat acc tgt ggt gaa 111 Pro Ile Cys His Thr Cys Gly Glu cag Gln gtg ggg cat gat Val Gly His Asp gca Ala 20 aat ggg gag cta Asn Gly Glu Leu gtg get tgc cat Val Ala Cys His gag Glu 158 206 tgt age tat ccc Cys Ser Tyr Pro tgc aag tct tgt Cys Lys Ser Cys gag ttt gaa atc Glu Phe Glu Ile aat gag Asn Glu ggc cgg aaa Gly Arg Lys ctg gat gat Leu Asp Asp tgc ttg cgg tgt Cys Leu Arg Cys ggc Gly tcg cca tat gat Ser Pro Tyr Asp gag aac ttg Glu Asn Leu aca atg gca Thr Met Ala gta gaa aag aag Val Glu Lys Lys ggg Gly 70 tct ggc aat caa Ser Gly Asn Gln tcc Ser tct cac Ser His ctc aac gat tct Leu Asn Asp Ser gat gtc gga atc Asp Val Gly Ile gct aga cat atc Ala Arg His Ile agt agt gtg tcc act gtg gat agt gaa atg aat gat gaa tat ggg aat Ser Ser Val Ser Thr Val Asp Ser Glu Met Asn Asp Glu Tyr Gly Asn 1 SUBSTITUTE SHEET (RULE 26) WO OOM670 WO 0071670PCTIU SOO/13 637 cca att tgg aag Pro Ile Trp Lys aat As n 115 cgg gtg aag agc Arg Val Lys Ser aag gat aaa gag Lys Asp Lys Giu aac aag Asn Lys 125 aag aaa aag Lys Lys Lys aca gaa cag Thr Giu Gin 145 aga Arg 130 agt cct aag gct Ser Pro Lys Ala gaa Giu 135 act gaa cca gct Thr Giu Pro Aia caa gtt cct Gin Val Pro 140 tcg gag ccg Ser Glu Pro 494 542 cag atg gaa gag Gin Met Giu Giu ccg tct gca gag Pro Sex Ala Giu gct Ala 155 ctt tca Leu Ser 160 att gtt tat cca Ile Vai Tyr Pro att Ile 165 cca cgc aac aag Pro Arq Asn Lys aca cca tac aga Thr Pro Tyr Arg qtg atc att atg Val Ile Ile Met ctg gtc att ctg Leu Val Ile Leu ggc Gly 185 ctc ttc ttc cac Leu Phe Phe His ttc Phe 190 590 638 686 aga ata aca aat Arg Ile Thr Asn cct Pro 195 gtc gat agt gcc Val Asp Ser Aia t tt Phe 200 ggc ctg tgg ctt Giy Leu Trp Leu act tct Thr Ser 205 gtc ata tgt Val Ile Cys ccc aag tgg Pro Lys Trp 225 atc tgg ttt gca Ile Trp Phe Ala tct tgg gtg ttg Ser Trp Vai Leu gat cag ttc Asp Gin Phe 220 agg ctg tcg Arg Leu Ser 734 782 aat cct gtc aat Asn Pro Vai Asn aga Arg 230 gaa acg tat atc Giu Thr Tyr Ile gaa Giu 235 gca agg Ala Arg 240 tat gaa aga gag Tyr Giu Arg Giu ggt Gly 245 gag cct tct cag Giu Pro Ser Gin ctt Leu 250 gct ggt gtg gat Ala Giy Vai Asp ttc gtg agt act Phe Val Ser Thr gt t Val1 260 gat ccg ctg aag Asp Pro Leu Lys gaa Giu 265 ccg cca ttg atc Pro Pro Leu Ile act Thr 270 830 878 926 gcc aat aca gtc Ala Asn Thr Val tcc atc ctt gct Ser Ile Leu Ala gac tat ccc gtc Asp Tyr Pro Vai gat aaa Asp Lys 285 gtc tcc tgc Val Ser Cys tct ctt gta Ser Leu Val 305 tac Tyr 290 gtg tct gat gat Vai Ser Asp Asp ggt Gly 295 gca gct atg ctt Ala Ala Met Leu tca ttt gaa Ser Phe Giu 300 ccg ttc tgc Pro Phe Cys 974 1022 gaa aca. gct gag Giu Thr Ala Giu gca agg aag tgg Ala Arg Lys Trp gtt Val1 315 aaa aaa.

Lys Lys 320 ttc tca att gaa Phe Ser Ilie Glu aga gca ccg gag Arg Ala Pro Giu ttt Phe 330 tac ttc tca cag Tyr Phe Ser Gin 1070 SUBSTITUTE SHEET (RULE 26) WO 00/71670 WO 0071670PCTIUSOO/1 3637 aaa att Lys Ile 335 cgt aga Arg Arg gcc ctg Ala Len caa gat Gin Asp cat gat His Asp 400 gga aat Gly Asn 415 ggc tac Gly Tyr gtg tct Val Ser gat cac Asp His ctg atg Leu Met 480 cag agg Gin Arg 495 gta gtt Val Val cca gta Pro Val ggc tac Gly Tyr gat Asp gca Ala gta Val1 gga Gly 385 tca Ser gaa Glu cag Gin gca Al a tat Tyr 465 gac Asp ttt Phe ttc Phe tac Tyr ggg Gly 545 tac Tyr atg Met gca Al a 370 aca Thr ggt Gly cta Leu cac His gt a Val1 450 gta Val1 cca Pro ga t Asp ttt Phe gta Val1 530 cct Pro ttg Leu aaa Lys 355 aag Lys cct Pro ctt Leu cct Pro cac His 435 ctc Leu aac As n caa Gin Gly gat Asp 515 gga Gly cct Pro aaa Lys 340 agg Arg gct Ala t gg Trp cct Pro cgt Arg 420 aaa Lys a ca Thr aat Asn gta Val ata Ile 500 gtt Val act Thr tct Ser ga c Asp gat Asp cag Gin cct Pro tgg Trp 405 cta Leu aag Lys aat Asn agc Ser ggt Gly 485 gat Asp aac Asn ggt Giy atg Met 3 aag Lys tat Tyr aaa Lys ggg Gly 390 gaa Giu gta Val1 gct Ala gct Ala aag Lys 470 cga Pirg aag Lys atg Miet tgt Dys ~c C Pro 550 gtt Val1 gaa Giu aca Thr 375 aat As n ata Ile tat Tyr ggt Gly ccc Pro 455 gct Ala gat Asp agt Ser aaa Lys gt t Val1 535 caa Gin gag Giu 360 cct Pro aac Asn ctg Len gt c Val1 gca Al a 440 tac Tyr gt t Val1 gt a Val1 gat Asp ggg Gly 520 ttc Phe cct Pro 345 tac Tyr gaa Giu aca Thr gga Gly tcc Ser 425 gaa Gin atc Ile cga Arg t gc Cys cgc Arg 505 ttg Leu aac Asn tct Ser aaa Lys gaa Gin cgt Arg gct Ala 410 agg Arg aat Asn ct c Len gag Gin tat Tyr 490 tac Tyr gat Asp a gg Arg ttc Phe gtc Val1 gga Gly ga t Asp 395 cgt Arg gag 6in gct Ala aat As n gca Ala 475 gtg Val1 gcc Ala ggc Gly caa Gin gtg Val1 cga Arg t gg Trp 380 cac His gac Asp aag Lys ctg Leu gtt Val 460 atg Met cag Gin aat Asn att Ile gca Ala 540 aaa Lys gt t Val1 365 act Thr cct Pro att Ile aga Arg gtg Val1 445 gat Asp tgc Cys ttc Phe cgt Arg caa Gln 525 ct t Len gaa Gin 350 aat Asn atg Met ggg Gly gaa Gin c ct Pro 430 aga Arg t gt Cys atc Ile cct Pro aa c Asn 510 gga Gly tac Tyr 1118 1166 1214 1262 1310 1358 1406 1454 1502 1550 1598 1646 1694 1742 agc tta cgc aag aga aag gat tct Ser Len Arg Lys Arg 555 Lys Asp Ser SUBSTITUTE SHEET- (RULE 26) WO oon]1670 PCTIUSOO/13637 tca tcc Ser Ser 560 tgc ttc tca tgt Cys Phe Ser Cys tgc ccc tca aag Cys Pro Ser Lys aag Lys 570 aag cct gct caa Lys Pro Ala Gin gat Asp 575 cca gct gag gta Pro Ala Giu Val tac Tyr 580 aga gat gca aaa Arg Asp Ala Lys aga Arg 585 gag gat ctc aat Glu Asp Leu Asn gct Ala 590 1790 1838 1886 gcc ata ttt aat Ala Ile Phe Asn aca gag att gat Thr Glu Ile Asp aat Asn 600 tat gac gag cat Tyr Asp Giu His gaa agg Glu Arg 605 tca atg ctg Ser Met Leu tct gtc ttc Ser Vai Phe 625 atc Ile 610 tcc cag ttg agc Ser Gin Leu Ser ttt Phe 615 gag aaa act ttt Glu Lys Thr Phe ggc tta tct Gly Leu Ser 620 gta ccc gag Val Pro Glu att gag tct aca Ile Giu Ser Thr atg gag aat gga Met Giu Asn Gly tct gcc Ser Ala 640 aac tca cca cca Asn Ser Pro Pro atc aag gaa gcg Ile Lys Giu Ala att Ile 650 caa gtc atc ggc Gin Val Ile Gly tgt Cys 655 ggc tat gaa gag Gly Tyr Giu Glu aag act Lys Thr 660 gaa tgg gga Glu Trp Gly aaa Lys 665 cag att ggt tgg Gin Ile Gly Trp 1934 1982 2030 2078 2126 2174 2222 tat ggg tca gtc Tyr Giy Ser Val gag gat atc tta Glu Asp Ile Leu ggc ttc aag atg Gly Phe Lys Met cac tgc His Cys 685 cga gga tgg Arg Giy Trp gga tct gca Gly Ser Ala 705 aga Arg 690 tca att tac tgc Ser Ile Tyr Cys atg Met 695 ccc gta agg cct Pro Val Arg Pro gca ttc aaa Ala Phe Lys 700 gtc ctc cga Val Leu Arg ccc atc aac ctg Pro Ile Asn Leu tct Ser 710 gat aga ttg cac Asp Arg Leu His cag Gin 715 tgg gct Trp Ala 720 ctt ggt tct gtg Leu Giy Ser Val att ttc ttt agc Ile Phe Phe Ser aga Arg 730 cac tgt ccc ctc His Cys Pro Leu tgg Trp 735 tac ggg ttt gga Tyr Giy Phe Gly gga Gly 740 ggc cgt ctt aaa Gly Arg Leu Lys tgg Trp 745 ctc caa agg ctt Leu Gin Arg Leu gcg Ala 750 2270 2318 2366 tat ata aac acc Tyr Ile Asn Thr att Ile 755 gtg tac cca ttt Val Tyr Pro Phe aca Thr 760 tcc ctc cct ctc Ser Leu Pro Leu att gcc Ile Ala 765 tat tgc aca Tyr Cys Thr cct gca gtt tgt Pro Ala Val Cys ctc acc gga aaa Leu Thr Gly Lys ttc atc ata Phe Ile Ile 780 2414 cca acg ctc tca aac ctg gca agc atg ctg ttt ctt ggc ctc ttt atc 4 2462 SUBSTITUTE SHEET (RULE 26) WO 00/71670 WO 0071670PCTIUSOOII3637 Pro tcc Ser a tt Ile 815 t ca Ser ggc Gly gaa Giu cca Pro ga t Asp 895 gtg Val ggt Gly tca Ser cca Pro tcc Ser 975 Thr atc Ile 800 gaa Giu gcc Ala at c Ile ttt Phe a cc Thr 880 gca Ala ttc Phe cta Leu gtg Val ttc Phe 960 att Ile Leu 785 att Ile gat Asp cat His gat Asp ggg Gly 865 a ca Thr ctc Leu ttt Phe atg Met ctg Le u 945 gtt Val1 gat Asp Ser Asn Leu gta act gcg Vai Thr Ala tta tgg cgt Leu Trp Arg 820 ctc ttt gcg Leu Phe Ala 835 acg aac ttc Thr Asn Phe 850 gag cta tat Giu Leu Tyr ctt ctc att Leu Leu Ile aac aaa. gga Asn Lys Giy 900 gct ttc tgg Ala Phe Trp 915 ggt cgc caa Gly Arg Gin 930 ttg gcc tct Leu Ala Ser aac aaa gtt Asn Lys Val tgc tgagctac Cys Ala gt g Val 805 aat As n gtc Val1 act Thr atg Met atc Ile 885 tat Tyr gtg Val aac Asn gtc Val gat Asp 965 Ser 790 ctt Leu ga a Giu ttc Phe gtc Val gtc Val 870 aat As n gaa.

Giu att Ile cta Leu ttc Phe 950 aac Met gag Giu caa Gin cag Gin aca.

Thr 855 aag Lys atg Met gca Ala ctt Le u aca Thr 935 tct Ser Leu Phe cta aga Leu Arg ttc tgg Phe Trp 825 gga ttc Gly Phe 840 gca aaa Ala Lys tgg aca Trp Thr tcg ggt Ser Gly tgg ggg Trp Gly 905 cat ctc His Leu 920 cca acc Pro Thr ctc gtt Leu Val Leu tgg Trp 810 gt g Val1 tta Leu gca Ala aca Thr tgt Cys 890 cct Pro tat Tyr att Ile tgg Trp, Gly 795 agc Se r atc Ile aaa Lys gcc Ala ctt Leu 875 gct Ala ctc Leu cca Pro gtt Val1 gtc Val1 955 gag Leu ggt Gly gga Gly atg Met ga a Giu 860 ttg Leu gga Gly ttt Phe ttc Phe gtt Val 940 aag Lys acc Phe gtc Val1 ggt Gly ttg Le u 845 ga t Asp at t Ile ttc Phe ggc Gly ctt Leu 925 ct c Leu atc Ile t gc Ile agc Ser gtt Val1 830 gct Ala gca Ala cct Pro tct Ser aag Lys 910 aaa Lys tgg Trp aat Asn att 2510 2558 2606 2654 2702 2750 2798 2846 2894 2942 2990 3042 acc ttg gtt gcg Asn Thr Leu Val Ala Giu Thr Cys Ile :.ct ccaataagtc tctcccagta ttttggggtt acaaaacctt tcgctgtcag tgaaatgata tgggaattgg aatatgatcc tcgttgtagt ttccctcaag aaagcacata tatttaaatg aactgcaaga tgattgttct ctatgaagtt ttgaacagtt ttatgttaaa atacaggttt tgattgtgtt gaaaaaaaaa aagaaaaaaa 3102 3162 3222 SUBSTITUTE SHEET (RULE 26) WO 00/71670 aaaaaaaaaa <210> 2 <211> 978 <212> PRT <213> Populus tremuloides PCTUSOO/1 3637 <400> 2 Met Met Glu Ser 1 Gly Tyr Lys Asp Leu Val Trp Lys Gin 145 Ile Ile Thr Cys Trp 225 Tyr His Pro Vai Vai Asn Ser Lys Arg 130 Gin Val Ile Asn Giu 210 Asn Giu Asp Met Cys Giu Asp Thr Asn 115 Ser Met Tyr Met Pro 195 Ile Pro Arg Ala Cys Leu Lys Ser Val1 100 Arg Pro Giu Pro Arg 180 Val Trp Val Glu Gly 5 As n Lys Arg Lys Gin Asp Val1 Lys Giu Ile 165 Leu Asp Phe Asn Gly 245 Gly Ser Cys Gly 70 Asp Ser Lys Aia Lys 150 Pro Val Ser Ala Arg 230 Glu Ala Pro Ile Cys His Thr Cys Gly Gl Giu Leu Cys Phe 40 Gly Ser 55 Ser Gly Val Gly Giu Met Ser Cys 120 Giu Thr 135 Pro Ser Arq Asn Ile Leu Ala Phe 200 Phe Ser 215 Giu Thr Pro Ser 6 Phe Giu Pro Asn Ile Asn 105 Lys Giu Ala Lys Giy 185 Gly Trp Tyr Gin Vali Phe Tyr Gin His Asp Asp Pro Giu Le u 170 Leu Leu Val1 Ile Leu 250 Ala Giu Asp Ser Ala Giu Lys Ala Al a 155 Thr Phe Trp Le u Glu 235 Ala Cys Ile Giu Thr Arg Tyr Giu Gin 140 Ser Pro Phe Leu Asp 220 Arg Gi y His As n Asn Met His Giy As n 125 Val1 Glu Tyr His Thr 205 Gin Leu Val Gl Gil Le, Ala Ly! Prc- Prc- Arc- Phe 1 9C Ser- Phe- Ser Asp SUBSTITUTE SHEET (RULE 26) WO OOn1670 WO 0071670PCT/tJSOO/13637 Val1 Thr Cys Val1 305 Phe Asp Ala Val Gly 385 Ser Glu Gin Ala Tyr 465 Asp Phe Phe Tyr Gly 545 Ser Val1 Tyr 290 Glu Ser Tyr Met Ala 370 Thr Gly Leu His Val 450 Val Pro Asp Phe Val 530 Pro Thr Leu 275 Val1 Thr Ile Le u Lys 355 Lys Pro Leu Pro His 435 Leu Asn Gin Gly Asp 515 Gly Pro Val1 260 Ser Ser Al a Glu Lys 340 Arg Ala Trp Pro Arg 420 Lys Thr Asn Val Ile 500 Val1 Thr Ser Asp Ile Asp Giu Pro 325 Asp Asp Gin Pro Trp 405 Leu Lys Asn Ser Gly 485 Asp Asn Gly Met Pro Le u Asp Phe 310 Arg Lys Tyr Lys Gly 390 Giu Val1 Ala Ala Lys 470 Arg Lys Met Cys Pro 550 Leu Ala Gly 295 Ala Ala Val Glu Thr 375 Asn Ile Tyr Gly Pro 455 Ala Asp Ser Lys Val1 535 Ser Lys Val1 280 Al a Arg Pro Gin Glu 360 Pro Asn Leu Val1 Ala 440 Tyr Val Val1 Asp Gly 520 Phe Leu Glu 265 Asp Ala Lys Glu Pro 345 Tyr Glu Thr Gly Ser 425 Glu Ile Arg Cys Arg 505 Le u Asn Arg Pro Tyr Met Trp, Phe 330 Ser Lys Giu Arg Ala 410 Arg Asn Leu Giu Tyr 490 Tyr Asp Arg Lys Pro Pro Le u Val1 315 Tyr Phe Val Gly Asp 395 Arg Glu Ala Asn Ala 475 Val Ala Gly Gin Arg 555 Leu Val1 Ser 300 Pro Phe Val Arg Trp 380 His Asp Lys Leu Val1 460 Met Gin Asn Ile Ala 540 Lys I le Asp 285 Phe Phe Ser Lys Val1 365 Thr Pro Ile Arg Val1 445 Asp Cys Phe Arg Gin 525 Leu Asp Thr 270 Lys Giu Cys Gin Giu 350 Asn Met Gly Giu Pro 430 Arg Cys Ile Pro As n 510 Gly Tyr Ser Ala Val1 Ser Lys Lys 335 Arq Ala Gin His Gly 415 Gly Val1 Asp Leu Gin 495 Vai Pro Gi y Ser As n Ser Leu Lys 320 Ile Arg Le u Asp Asp 400 As n Tyr Ser His Met 480 Arg Val1 Val1 Tyr Ser 560 SUBSTITUTE SHEET (RULE 26) WO 00/71670 PCT/US00/13637 Cys Phe Ser Cys Cys Cys Pro Ser Lys Lys Lys Pro Ala Gin Asp Pro 565 570 575 Ala Glu Val Tyr Arg Asp Ala Lys Arg Glu Asp Leu Asn Ala Ala Ile 580 585 590 Phe Asn Leu Thr Glu Ile Asp Asn Tyr Asp Glu His Glu Arg Ser Met 595 600 605 Leu Ile Ser Gin Leu Ser Phe Glu Lys Thr Phe Gly Leu Ser Ser Val 610 615 620 Phe Ile Glu Ser Thr Leu Met Glu Asn Gly Gly Val Pro Glu Ser Ala 625 630 635 640 Asn Ser Pro Pro Phe Ile Lys Glu Ala Ile Gin Val Ile Gly Cys Gly 645 650 655 Tyr Glu Glu Lys Thr Glu Trp Gly Lys Gin Ile Gly Trp Ile Tyr Gly 660 665 670 Ser Val Thr Glu Asp Ile Leu Ser Gly Phe Lys Met His Cys Arg Gly 675 680 685 Trp Arg Ser Ile Tyr Cys Met Pro Val Arg Pro Ala Phe Lys Gly Ser 690 695 700 Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gin Val Leu Arg Trp Ala 705 710 715 720 Leu Gly Ser Val Glu Ile Phe Phe Ser Arg His Cys Pro Leu Trp Tyr 725 730 735 Gly Phe Gly Gly Gly Arg Leu Lys Trp Leu Gin Arg Leu Ala Tyr Ile 740 745 750 Asn Thr Ile Val Tyr Pro Phe Thr Ser Leu Pro Leu Ile Ala Tyr Cys 755 760 765 Thr Ile Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr 770 775 780 Leu Ser Asn Leu Ala Ser Met Leu Phe Leu Gly Leu Phe Ile Ser Ile 785 790 795 800 Ile Val Thr Ala Val Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Glu 805 810 815 Asp Leu Trp Arg Asn Glu Gin Phe Trp Val Ile Gly Gly Val Ser Ala 820 825 830 His Leu Phe Ala Val Phe Gin Gly Phe Leu Lys Met Leu Ala Gly Ile 835 840 845 Asp Thr Asn Phe Thr Val Thr Ala Lys Ala Ala Glu Asp Ala Glu Phe 850 855 860 8 SUBSTITUTE SHEET (RULE 26) WO 00/71670 WO 0071670PCT/USOO/13637 Gly 865 Thr Glu Leu Tyr Met Val1 870 Asn Lys Trp Thr Thr Leu Leu Ile Ile 885 Leu Asn Lys Phe Ala Phe 915 Met Gly Arg Gly 900 Trp Tyr Giu Vai Ile Met Ser Gly Ala Trp Gly 905 Leu His Leu Cys 890 Pro Leu Leu Ile 875 Ala Gly Phe Leu Phe Gly Pro Pro Ser Asp Ala 895 Lys Val Phe 910 Lys Gly Leu Trp Ser Val Thr 880 Tyr Pro Phe 920 Thr Pro Leu 925 Leu Gin Asn Leu Thr Ile Val 930 Leu Leu Ala Ser Val 945 Val1 Phe 950 As n Leu Val Trp Val1 955 Glu Ile Asn Pro Asn Lys Val Asp 965 Thr Leu Val Ala 970 Thr Cys Ile Ser 975 Asp Cys <210> <211> <212> <213> <220> <221> <222> <220> <223> 3 1010

DNA

Populus tremuloides

CDS

(1008) 5' flanking region of PtCelA coding sequence <400> 3 gaattcgccc cttctggtct caagagatgg atagtctgat tgttaatcct gagatctcca acattgacat ggatcctgcc taatgacttg ttttgaattc agcaatttgc gttctatggt tcgaagttgc gtagctatta ttctacgttt gatgattgat agttttcagt tattgaaagt aggagacgat aaaagaagtt cacttattta aaactgccgt gcggaccaac ctttctaatt gattatggga tcacatggca ttggtaagtt 9 agtttccggt acaaaacaaa tgcccatcat ttctggtatt aaccagatat tttccgtttc accattccga tctcagccca gaagatgtgc tcgttgaatg tgcatattat ttgttctggg gcaattatgt acgggatcag agtgagagaa tgttagacac agatcatgtg tctgcccaac gctttgttca gtaaatttaa gttactcttt agccataaac cgtcgtaaaa ttaccctgat gagaccatct tttatacgcc agaaaccttc SUBSTITUTE SHEET (RULE 26) wo oonivo WO 0071670PCT/USOO/1 3637 cttaaatttc acttgggttc cttctcttcg gctttggtta aggaagaccc atg atg gaa Met Met Glu 1 ggg cat gat Giy His Asp tat ccc atg Tyr Pro Met aaa gtt tgc Lys Vai Cys cagcaaatct ttcaaacttg gccttacacc ttggaaacca tgcaccaacc gccatacccc ccattacaaa aatgtcagta ccaccctctg gggtatttga tataaaaaca aggccaaaac tcttgaaaga attgaagttg taaagagctg tct qgg gct cct ata tgc cat acc Ser Giy Ala Pro Ile Cys His Thr 5 10 gca aat ggg gag cta ttt gtg gct Ala Asn Giy Giu Leu Phe Val Ala 25 tgc aag tct tgt ttc gag ttt gaa Cys Lys Ser Cys Phe Giu Phe Glu ccgaaaatag accaacccac aaagacacca aaaagattgg gtaaagtggt tgt ggt gaz Cys Gly Gl.

tgc cat gac Cys His Gli 3C atc aaa gac Ile Lys GlL acgtgcttct caccctcaac acacacccta aaggaagcag aataagcaag kcag gtg iGin Vai 1tgt agc iCys Ser 600 660 720 780 840 888 936 ggc cgg 984 Gly Arg tcg ag Ser ttg cgg tgt Leu Arg Cys ggc Giy 1010 <210> 4 <211> 56 <212> PRT <213> Populus tremuloides <223> 5' fianking region of <400> 4 Met Met Giu Ser Giy Aia Pro 1 5 Gly His Asp Ala Asn Gly Giu PtCeiA coding sequence Ile Leu Cys His Thr Cys Giy Giu Gin Vai 10 Phe Val Ala Cys His Glu Cys Ser 25 Giu Phe Glu Ile Lys Glu Gly Arg Cys Tyr Pro Met Lys Vai Cys Lys Ser Cys Leu Arg Cys Gly <210> <211> 3444 <212> DNA <213> Arabidopsis thaliana <220> <223> cellulose synthase mRNA <400> SUBSTITUTE SHEET (RULE 26) WO 00/71670 WO 0071670PCT/USOO/13637 gcggccgcgg tgagaatgcc agatgagatc ccctgtgtgt gtgcaaaacc agatgacatt tcaggtttct ggattcagct gatttcttct agttcatcct tcagaaagat atggaagaga tgaagatggt gaagatacca t gt gattct t tttgtqqctt gttccctaaa tgagaaagaa tccattgaaa ttatcctgtc cgaagctctt ttgtattgaq taaagttcat caaaqtaaag tatgcaagac ggtcttcctt ttacgtttct ttccctgata tgatcactac tcagtcagga gcacgat cga tgqgctacaa cggatttgat atggtgtctc taagaagaag gggcCgCggt gcagaagaaa gatggctaga cggatatgaa cgaaqatatt accaaagtta agttcttcga gtat ggt tat ttacccgtgg cactggaaaa cttctcgtcg tgattqgtgg tctcttccaa aaaagcagct catccctcca tgccatcagc ttgggtcatc gccaaccatt ccqggttaat cttgtgattc agagaggtaa gatgaaaaat ttaatcgccg cgaataagat gaattgactg agaccttgct cgtttcaaac gatgatttag gaaggtatgt ccacctqgct gatagacatg gt tt ctctt t cttgcggttt aagcagaatg gatgatgctg atcaaatcga ggtctcttct atttctgtta tggtacccta gggaaaccgt gagcctccgc gataaggttg tctgagaccg cctcgtgctc cccgcatttg atcaatgctt ggtacacctt ggaagtgacg cqtgagaaga cgagtctctg atcaacaata aagaaaatct tactcaaatc gggcctatat gcaccgaaga ctatgttttg aaqaataggg cataaagttc tatgggcagt aacgcaagcc gataaaactg cttacgggtt gcggctttca tqggcgcttg ggagqtgggt acctctctac ttcatcgttc attgcaataa a gaa acgaa c ggtctcctca gatgatggag a tgactctac aatggatacg attcatcttt attgtcgtct ccgtttgtgg gattgaccgg gagagatatt gaaaagaaaa gttctcacaa cagtccaaga ttgatggaga atgagtacga gtcttaaagg acaatgagtt caatctctcq ctcagattcc ctcttattgt ctqacccgac atggttatgg agaaacttca attttccaat gcaagataaa ttcactaccg tatgtgagat tcgagcgaqa cgggactatc ttattactgc cttqttacgt ctgaattcqc ccgaatggta ttaggqagcq tagtagcaac gqcccgqtaa gtgttcgtga gacccggatt gggttctatc gcaaagctct gttatgttca gcaatgttgt acgtcggtac agaagaagqq qttcaagaaa aagcgtcaaa ttaacgtaga ctcctgtatt cggcttgtct aatggggaaa ctaagatgca aaggatcagc ggtcggttga tgaaatgqct cgctcatcgt ccgagattag cgggtattct agttttggqt aqgttcttgc agttctctga tcatcataaa actcgtgggg acccgttcct ggtccatcct cgaaaqgcgg tggatgggtt gttttacctc agaagattta caggaatgag gctgagtgga accgtttgtg aagacgaqaa aagtccaaga tqagtatgga tcgcaactcc attgctqact tcctccttca cgtggctgca aagtgtcqct ggttgttag gatggatgag tccttaccgg tattcttcac atggtttgct aacgtacttg ccctgtggat aaatactgtc atctgatgat aaggaaatgg tttctgccat gcgagccatg agcacagaaa taqtgtgcga tgtcgaaaac tgatcaccat aaatgctcct tagagaagca gttccctcaa gttctttqat aggttgtgtt cccacqtaag qaatcgtaaa gcagatccac acagtcaacc tgttgcatct gcttaaagaa agagattggg ttctcatggt tccaatcaat gattttcttg tgagcggttg ttactgttct caactatgcg cgagatgcaa cattggaggt tggtgtcgac cctttacctc cgtcattgga accgcttttc taaaggtttg cctggcct cg tcctattctc ggtgaaaaag taaaagactc attttgttac tttgtcctca cagacatgtc gcatgtaacg ggcaatcaag gttgaaggtg aataatggga ggtttcccac tacggcgacg cttggtggtc catcgaaggc tqgaaaqatc catgaaggag ggaaggcagc atgttaattg cccgtcaaag gtttcatggg qaccgactct gtatttgtta ttgtctattc ggtgctgcta gttcctttct aaaatggact aagagagatt gtgcctgagg gatcatcctg aacgagttgc aagaaggct g taccttctga atgtgtttca aggttcgatg atcaatatga ttcagqaggc acatgcaatt gcaaagacag gcattagaaa gaggcaatgc gcgcgtctgg gccatccaag tqgatctatg tggagacatg ctttcggatc agtaggcatt tcctacatta ctccctgcca agtatcctct tggggcaaag gtttctgcgc actaacttca ttcaaatgga gtcatagtcg ggaaqactgt cttgggaaac attcttacac gagatctgtg gtttaattcc cttcattgtg gagaattgtt ttaatgccga aaatctgcag aatgtgcatt cttgtccaca atgaagagga ttggatttga aatctgattt aggacgttga atgqcaatag tgatggtacc ggatgqagga atcctqattt cattgtctat tgctacgtct atgcatatgc ttcttgatca cattaagata gtacagtgga ttgctgttga tgcttacttt gcaagaaata acttgaagaa atgaagaatt atggttggac gcatgattca ctcgattagt gaqctatgaa atgtcgattg tgatggatcc ggattgatag aaggtttqqa aagcgcttta gctgqccaaa tggctgcgga atatcgaaga aaatgaagtt agaatggtgg tcattagtcg gttctqttac tttattgtac gtctccatca gtcctatttg actctgtggt tctgtcttct tcatggcgct ttgggatcga atctgtttgc cagtcacatc cttcacttct qa gtct ct ga tctttgcact aagatagaat ttctttgggt gtttagactg cacgqatcaa ttcattagat atttttgcaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 SUBSTITUTE SHEET (RULE 26) WO 00/71670 gaatgtgttg tagatagcgg ccgc <210> 6 <211> 1080 <212> PRT <213> Arabidopsis thaliana <220> <223> cellulose synthase PCT/USOO/13637 3444 <400> 6 Arg Pro 1 Ile Asn Gly Gin Gly Giu Pro Cys Cys Lys Asp Giu Gly Asn Ser Arq 130 Pro Gly 145 Ile Ser His Gly Ala His Tyr Giy 210 Gin Asn 225 Arg Ala Thr Pro Tyr Thr Giu As n 115 Arg Ser Ser As n Arg 195 Ser Giu Ile 5 Giu Gin Val1 Tyr Phe Asp Ile Ser Ile Arg 165 Val1 Leu Ala Leu Ala As n Ile Al a Giu 70 Lys Asp Gly Gly Pro 150 His His Met Trp Gin 230 Gi y Ala Cys Cys 55 Arq Arg Ile Phe Phe 135 Leu Al a Pro Val Lys 215 Val1 Ser Arq Arg 40 As n Arg Leu Asp Asp 120 Pro Leu Leu Val1 Pro 200 Asp Val1 12 His Ile Asp Giu Glu Lys Asp 105 Gin Gin Thr Ile Ser 185 Gin Arg Arg As n 10 Arg Giu Cys Gly Gi y 90 Leu Val1 Ser Tyr Val1 170 Leu Lys Met His Arg Ser Ile Ala Asn 75 Ser Asp Ser Asp Gly 155 Pro Ser Asp Glu Giu 235 Asn Val1 Giu Phe Gin Pro As n Glu Leu 140 Asp Pro Asp Leu Giu 220 Gly Giu Gin Leu Pro Ala Arg Giu Gly 125 Asp Giu Ser Pro Ala 205 Trp Asp Phe Giu Thr Val Cys Val Phe 110 Met Ser Asp Leu Thr 190 Val Lys Pro Val1 Leu Val Cys Pro Giu Giu Ser Al a Val1 Gly 175 Val Tyr Arg Asp Leu Ser Asp Arg Gin Gi y Tyr Ile Pro Giu 160 Gi y Ala Gi y Lys Phe 240 SUBSTITUTE SHEET (RULE 26) WO 00171670 WO 0071670PCT/USOO/1 3637 Giu Pro Arg Tyr Ser 305 Phe Ser Asp Thr Lys 385 Giu Cys His Glu Asn 465 Met Gly Asn Gly Asp Leu Met Arg 290 Val1 Pro Leu Val1 Ala 370 Val1 Ala Lys Lys Arq 450 Ala Gin Met Asn Phe 530 Asp Met 260 Ile Leu Cys Trp Tyr 340 Val1 Thr Cys Ser Tyr 420 Asp Ala Val1 Gly Gin 500 Leu His Asp 245 Lys Val1 His Giu Tyr 325 Giu Ser Val1 Tyr Glu 405 Cys Tyr Met Ala Thr 485 Vai Pro His Ala Ile Le u Pro Ile 310 Pro Lys Thr Le u Vai 390 Thr Ile Leu Lys Thr 470 Pro Phe Arg Lys Asp Pro Arg Val1 295 Trp Ile Giu Val Ser 375 Ser Ala Glu Lys Arg 455 Ala Trp Leu Leu Lys 535 Phe Ile Leu 280 Lys Phe Giu Gly Asp 360 Ile Asp Giu Pro Asn 440 Asp Gin Pro Gly Val1 520 Al a Pro Lys 265 Val Asp Ala Arg Lys 345 Pro Le u Asp Phe Arg 425 Lys Tyr Lys Gly Ser 505 Tyr Gly Met 250 Ser Ile Ala Val1 Glu 330 Pro Leu Ala Gly Ala 410 Al a Val Glu Val1 Asn 490 Asp Val Ala Met Ser Leu Tyr Ser 315 Thr Ser Lys Val1 Ala 395 Arg Pro His Glu Pro 475 Ser Gi y Ser Met Asp Lys Gly Ala 300 Trp Tyr Gly Glu Asp 380 Ala Lys Glu Pro Phe 460 Glu Val1 Val1 Arq Asn 540 Glu Ile Leu 285 Le u Val Le u Leu Pro 365 Tyr Met Trp Trp Ala 445 Lys Asp Arg Arq Glu 525 Ser Gly Asn 270 Phe Trp Leu Asp Ser 350 Pro Pro Leu Val Tyr 430 Phe Val1 Gly Asp Asp 510 Lys Le u Arg 255 Pro Phe Leu Asp Arg 335 Pro Leu Val1 Thr Pro 415 Phe Val Lys Trp His 495 Val1 Arg I le Gin Tyr His I le Gin 320 Leu Val Ile Asp Phe 400 Phe Cys Arg Ile Thr 480 Pro Glu Pro Arg SUBSTITUTE SHEET (RULE 26) WO 00/71670 PCriUSOOii 3637 Val1 545 Asp Met Gin Val Pro 625 Phe Trp Ala Lys Val1 705 Lys Asn Ala Lys Gi y 785 Lys Leu Ser Ser His Met Arg Val 610 Ile Asp Pro Lys Gin 690 Leu Lys Giy Ile Giu 770 Ser Le u His Arg Giy Tyr Asp Phe 595 Phe Tyr Ala Lys Thr 675 Ile Asn Tyr Gly Gin 755 Ile Lys Ala Gin His 835 Val Leu Ser 550 Ile Asn Asn 565 Pro Gin Ser 580 Asp Gly Ile Phe Asp Ile Val Thr Gly 630 Pro Lys Lys 645 Trp Cys Leu 660 Val Ala Ala His Ala Leu Val Giu Gin 710 Gly Gin Ser 725 Met Ala Arg 740 Vai Ile Ser Giy Trp Ile Met His Ser 790 Ala Phe Lys 805 Vai Leu Arg 820 Cys Pro Ile Asn Aia Pro Tyr Leu Leu Asn Vai Asp Cys 555 560 Ser Giy Asp Asn 615 Cys Lys Leu Asp Glu 695 Ser Pro As n Arg Tyr 775 His Gi y Trp Trp Lys Lys Arg 600 Met Val1 Lys Cys Lys 680 As n Thr Val1 Ala Giy 760 Gly Gly Ser Al a Tyr 840 14 Ala Lys 585 His Lys Phe Gly Phe 665 Lys Ile Giu Phe Ser 745 Tyr Ser Trp Ala Leu 825 Gly Leu 570 Ile Asp Giy Arg Pro 650 Gly Lys Glu Ala Vai 730 Pro Giu Val Arg Pro 810 Gly Tyr Arg Cys Arg Leu Arg 635 Arg Ser Lys Giu Met 715 Ala Ala Asp Thr His 795 Ile Ser Giy Giu Tyr Tyr Asp 620 Gin Lys Arg Asn Gly 700 Gin Ser Cys Lys Giu 780 Val Asn Val1 Gi y Ala Val Ser 605 Gly Ala Thr Lys Arg 685 Arg Met Ala Leu Thr 765 Asp Tyr Leu Giu Giy 845 Met Gin 590 As n Leu Le u Cys As n 670 Giu Gly Lys Arg Leu 750 Giu Ile Cys Ser Ile 830 Leu Cys 575 Phe Arg Gin Tyr Asn 655 Arg Aia His Leu Leu 735 Lys Trp Le u Thr Asp 815 Phe Lys Phe Pro As n Giy Giy 640 Cys Lys Ser Lys Gin 720 Glu Giu Giy Thr Pro 800 Arg Leu Trp SUBSTITUTE SKEET (RULE 26) .1 WO OOn1670 WOOO/1670PCT/USOO/13637 Leu Giu 850 Leu Pro 865 Gly Lys Met Ala Trp Gly Vai Ile 930 Leu Lys 945 Ala Ala Ser Leu Val Ie Gly Pro 1010 Leu Tyr 1025 Thr Ile Arg Le u Phe Leu Lys 915 Gly Val1 Asp Leu Val1 995 Leu Pro Ile Leu Ile Ile Phe 900 Val1 Gly Leu Asp Ile 980 Gly Phe Phe Val1 Ser Tyr Ile Asn 855 Val Tyr Cys Ser 870 Val Pro Giu Ile 885 Ser Ser Ile Ala Gly Ile Asp Asp 920 Val Ser Ala His 935 Ala Gly Val Asp 950 Giy Glu Phe Ser 965 Pro Pro Met Thr Val Ser Asp Ala 1000 *Gly Arg Leu Phe 1015 *Leu Lys Gly Leu 1030 Val Trp Ser Ile 1045 Vai Asn Pro Phe Ser Val Val Tyr Pro Trp Thr Ser 860 Leu Pro Ala Ile Cys Leu Leu Thr 875 880 Ser Asn Tyr Ala Ser Ile Leu Phe 890 895 Ile Thr Gly Ile Leu Giu Met Gin 905 910 Trp Trp Arg Asn Giu Gin Phe Trp 925 Leu Phe Ala Leu Phe Gin Giy Leu 940 Thr Asn Phe Thr Val Thr Ser Lys 955 960 Asp Leu Tyr Leu Phe Lys Trp Thr 970 975 Leu Leu Ile Ile Asn Val Ile Gly 985 990 Ile Ser Asn Gly Tyr Asp Ser Trp 1005 Phe Ala Leu Trp Vai Ile Ile His 1020 Leu Giy Lys Gin Asp Arg Met Pro 1035 1040 Leu Leu Ala Ser Ile Leu Thr Leu 1050 1055 Val Ala Lys Gly Giy Pro Ile Leu 1065 1070 Leu Trp Val *Arg 1060 Giu Ile Cys Gly Leu Asp Cys Leu 1075 1080 SUBSTITUTE SHEET (RULE 26)

Claims (2)

13- 4-05;l:27AM;MSJ Mel ,+61 2 9296 3999 5/ WHAT IS CLAIMED IS: 1 A plant transgenic for a polynucleotide encoding a polypeptide having at least identity with SEQ ID NO:2. 2 An insolated polynucleotide comprising SEQ ID NO:3. 3 A vector comprising the polynudeotide of claim 2. 4 A plant transgenic for the polynucleotide of claim 2. A method of altering the growth of a plant, comprising expressing in cells of the plant an exogenous polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:2 wherein the polynucleotide is expressed in an amount cffective to alter the growth of the plant. 6 A method according to claim 5, wherein the polynucleotide encodes SEQ ID NO:2. S. 7 A method according to claim 5, wherein the polynucleotide is in a sense 1 orientation. 8 A method according to claim 5, wherein the polynucleotide is in an antisense S. orientation. 9 A method according to claim 5, wherein a plant promoter, or a transcription factor binding domain thereof, is operatively linked to the polynucleotide. 9 10 A method according to claim 9, wherein the promoter is selected from S. constitutive promoters, tissue-specific promoters and developmental-specific plant promoters. 11 A method according to claim 10, wherein the promoter is Cauliflower Mosaic Virus 35S, 4CL, cellulose synthase promoter, PtCelAP or terminal flower promoter. 12 A method of altering the growth of a plant, comprising expressing an exogenous polynucleotide encoding a polypeptide comprising amino acid residues from position 220 to position 749 of SEQ ID NO:2 wherein expression of the polynucleotide alters the growth of the plant. 13 A method according to claim 12, wherein the polynuclcotide is in a sense orientation.

7718456.01 COMS ID No: SBMI-01203018 Received by IP Australia: Time 11:31 Date 2005-04-13 -36- 14 A method according to claim 12, wherein the polynucleotide is in a antisense orientation. A method of altering the cellulose content in a plant comprising expressing an exogenous polynucleotide encoding a polypeptide comprising amino acid residues from position 220 to position 749 of SEQ ID NO: 2 in a plant genome to alter the cellulose content of the plant. 16 A transgenic plant having an increased ratio of cellulose to lignin in cells of the plant, the plant comprising an exogenous polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:2 operably linked to a promoter so that the polynucleotide is expressed in an amount effective to increase the cellulose content of the plant. 17 The plant of the claim 16 wherein the polynucleotide encodes SEQ ID NO:2. 18 A method of altering expression of a cellulose synthase in a plant cell comprising 9. delivering into the cell a vector comprising a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:2. o 19 The method according to claim 18, wherein the polynucleotide encodes SEQ ID NO:2. 20 The method according to claim 18, wherein the polynucleotide is in a sense o* orientation. 21 The method according to claim 18, wherein the polynucleotide is in a antisense orientation. *9 9 22 A method of causing stress-induced gene expression in a plant cell comprising delivering into the cell a vector comprising SEQ ID NO:3 operatively linked to a gene, wherein the gene is expressed upon a mechanical stress to the plant. 23 A method of determining a positive mechanical stress responsive element (MSRE) in a cellulose synthase promoter comprising; introducing into a plant a cellulose synthase promoter that has a portion deleted, the cellulose synthase promoter operatively linked to a polynucleotide encoding a reporter, and (ii) detecting a decrease in the amount of reporter in the plant after inducing a stress to the plant. 24 A method of determining a negative MSRE in a cellulose synthase promoter comprising: -37- introducing into a plant a cellulose synthase promoter that has a portion deleted, the cellulose synthase promoter operatively linked to a reporter gene, and (ii) detecting an increase in the amount of reporter in the plant after inducing a stress to the plant. A method of expressing cellulose synthase in a plant in a tissue-specific manner comprising transforming the plant with a tissue-specific promoter operatively linked to a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:2. 26 A plant transgenic for a polynucleotide comprising a promoter and encoding a polypeptide having at least 95% identity to SEQ ID NO:2, the polynucleotide expressed such that the growth of the plant is altered relative to a similar control plant that does not contain the polynucleotide. 27 A method of altering a characteristic of a plant comprising genetically upregulating a polypeptide having at least 95% identity to SEQ ID NO:2 in the plant, wherein the characteristic is accelerated growth, increased cellulose content or decreased lignin content. 0 28 The method of claim 27 wherein the polynucleotide encodes SEQ ID NO:2. *t 0 0 29 A method of altering cellulose content in a plant comprising: delivery into cells of the plant an expression cassette comprising a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO:2 operably s linked to a promoter functional in a plant cell; and o0* (ii) expressing the polynucleotide in the cells of the plant in an amount effective to alter the cellulose content in the cells of the plant. A transgenic plant cell comprising an exogenous polynucleotide encoding a polypeptide consisting essentially of amino acid residues from position 220 to position 749 of SEQ ID NO:2, wherein the polypeptide has cellulose synthse activity. 31 A transgenic plant comprising the plant cell of claim 32 The transgenic plant of claim 31, wherein the plant is a tree. 33 A construct comprising an isolated polynucleotide encoding a polypeptide consisting essentially of amino acid residues from position 220 to position 749 of SEQ ID NO:2, wherein the polypeptide has cellulose synthase activity and wherein the polynucleotide is operably associated with a promoter sequence functional in a plant. th 0 -38- 34 A method of producing a transgenic plant cell comprising introducing into the plant cell an exogenous polynucleotide encoding a polypeptide consisting essentially of amino acid residues from position 220 to position 749 of SEQ ID NO:2. Dated this 6th day of April 2005 The Board of Control of Michigan Technological University by its patent attorneys Mallesons Stephen Jaques too. 0. 0* G *Gob